Anti-pro/latent-myostatin antibodies and uses thereof

ABSTRACT

Aspects of the present disclosure relate to antibodies that specifically bind proMyostatin and/or latent Myostatin and uses thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/760,393, filed Mar. 15, 2018, which is a 35 U.S.C. § 371 nationalstage filing of International Application No. PCT/US2016/052014, filedSep. 15, 2016, and claims priority under 35 U.S.C. § 119(e) of U.S.Provisional Patent Application No. 62/219,094, filed Sep. 15, 2015, andentitled “ANTI-PRO/LATENT-MYOSTATIN ANTIBODIES AND USES THEREOF”. Theentire contents of each of the foregoing applications are incorporatedherein by reference for all purposes.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 7, 2018, isnamed 127036-00820_SL.txt and is 52,594 bytes in size.

FIELD OF THE DISCLOSURE

Embodiments of the present disclosure may include modulators of growthfactor activity. In some embodiments, such modulators may includeantibodies and may modulate TGF-β family member activity and/or biology.

BACKGROUND OF THE DISCLOSURE

Myostatin is a secreted growth factor which negatively regulates musclemass. Loss of function mutations in the Myostatin gene, leading to ahypermuscular phenotype, have been described in cattle, sheep, fish,dogs and humans. Myostatin expression is generally limited to skeletalmuscle, with low levels of expression reported in adipose and cardiactissues. Inhibition of Myostatin signaling leads to an increase inmuscle size.

SUMMARY OF THE DISCLOSURE

Aspects of the disclosure relate, in some embodiments, to antibodiesthat bind specifically to forms of Myostatin (e.g., proMyostatin and/orlatent Myostatin). For example, antibodies provided herein specificallybind to one or more of a pro-form, and/or a latent-form of Myostatin,such as proMyostatin and/or latent Myostatin. In certain aspects, thedisclosure is based on the surprising discovery of antibodies providedherein that specifically bind pure, or substantially pure, proGDF8 (alsoreferred to as proMyostatin). In some embodiments, antibodies providedherein inhibit Myostatin signaling. In some embodiments, inhibition ofMyostatin signaling is useful for increasing muscle mass or preventingmuscle atrophy. In some embodiments, antibodies provided herein bind toand prevent cleavage of Myostatin by a proprotein convertase and/or atolloid protease. Preventing cleavage of proMyostatin or latentMyostatin, in some embodiments, prevents Myostatin activation. Furtheraspects of the disclosure relate to antibodies having an affinity to anantigen that is sensitive to pH. In some embodiments, such pH sensitiveantibodies are effective for clearing antigens from serum. Furthermore,in some embodiments, antibodies provided herein are sweeping antibodiesthat can efficiently clear antigens (e.g., proMyostatin and/or latentMyostatin) from serum.

Aspects of the present disclosure include an antibody that comprises aheavy chain variable domain and a light chain variable domain, in whichthe heavy chain variable domain comprises a complementarity determiningregion 3 (CDRH3) comprising a sequence as set forth in any one of SEQ IDNOs: 10-11. In some embodiments, an antibody specifically binds topro/latent-Myostatin. In some embodiments, the light chain variabledomain comprises a complementarity determining region 3 (CDRL3)comprising a sequence as set forth in any one of SEQ ID NO: 22-23. Inanother embodiment, said antibody comprises six complementaritydetermining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, andCDRL3, wherein CDRH1 comprises a sequence as set forth in any one of SEQID NOs: 1-3, CDRH2 comprises a sequence as set forth in any one of SEQID NOs: 4-9, CDRH3 comprises a sequence as set forth in any one of SEQID NOs: 10-11, CDRL1 comprises a sequence as set forth in any one of SEQID NOs: 12-17, CDRL2 comprises a sequence as set forth in any one of SEQID NOs: 18-21, and CDRL3 comprises a sequence as set forth in any one ofSEQ ID NOs: 22-23.

In some embodiments, said CDRH1 comprises a sequence as set forth in SEQID NO: 1 or 2, CDRH2 comprises a sequence as set forth in SEQ ID NO: 4or 5, CDRH3 comprises a sequence as set forth in SEQ ID NO: 10, CDRL1comprises a sequence as set forth in SEQ ID NO: 12 or 13, CDRL2comprises a sequence as set forth in SEQ ID NO: 18 or 19, and CDRL3comprises a sequence as set forth in SEQ ID NO: 22.

In another embodiment, said CDRH1 comprises a sequence as set forth inSEQ ID NO: 1 or 3, CDRH2 comprises a sequence as set forth in SEQ ID NO:6 or 7, CDRH3 comprises a sequence as set forth in SEQ ID NO: 11, CDRL1comprises a sequence as set forth in SEQ ID NO: 14 or 15, CDRL2comprises a sequence as set forth in SEQ ID NO: 20 or 21, and CDRL3comprises a sequence as set forth in SEQ ID NO: 23.

In other embodiments, CDRH1 comprises a sequence as set forth in SEQ IDNO: 1 or 3, CDRH2 comprises a sequence as set forth in SEQ ID NO: 8 or9, CDRH3 comprises a sequence as set forth in SEQ ID NO: 11, CDRL1comprises a sequence as set forth in SEQ ID NO: 16 or 17, CDRL2comprises a sequence as set forth in SEQ ID NO: 20 or 21, and CDRL3comprises a sequence as set forth in SEQ ID NO: 23.

In another embodiment, said antibody comprises a heavy chain variabledomain sequence as set forth in any one of SEQ ID NOs: 25-29. In someembodiments, said antibody comprises a light chain variable domainsequence of as set forth in any one of SEQ ID NOs: 30-35.

Other aspects of the disclosure include an antibody that specificallybinds to pro/latent-Myostatin and that comprises a heavy chain variabledomain and a light chain variable domain, in which the light chainvariable domain comprises a complementarity determining region 3 (CDRL3)comprising a sequence as set forth in any one of SEQ ID NO: 22-23. Insome embodiments, said antibody comprises a light chain variable domainsequence of SEQ ID NO: 30.

Some aspects of the disclosure relate to a polypeptide having a sequenceselected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 25, SEQID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO 29. In someembodiments, the polypeptide is a variable heavy chain domain. In someembodiments, the polypeptide is at least 75% (e.g., 80%, 85%, 90%, 95%,98%, or 99%) identical to any one of the amino acid sequences set forthin SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ IDNO: 28, or SEQ ID NO 29.

Some aspects of the disclosure relate to a polypeptide having a sequenceselected from the group consisting of SEQ ID NO: 30, SEQ ID NO: 31, SEQID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO 35. In someembodiments, the polypeptide is a variable light chain domain. In someembodiments, the polypeptide is at least 75% (e.g., 80%, 85%, 90%, 95%,98%, or 99%) identical to any one of the amino acid sequences set forthin SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ IDNO: 34, or SEQ ID NO 35.

Another aspect of the disclosure includes an antibody that competes forbinding to pro/latent-Myostatin with an antibody described above. Insome embodiments, said antibody binds to pro/latent-Myostatin at thesame epitope as an antibody described above. In another embodiment, anantibody competes for binding to pro/latent-Myostatin with anequilibrium dissociation constant, Kd, between the antibody andpro/latent-Myostatin of is less than 10⁻⁶ M. In other embodiments, saidantibody's Kd is in a range of 10⁻¹¹ M to 10⁻⁶ M.

In some embodiments, an antibody is a humanized antibody, a diabody, achimeric antibody, a Fab fragment, a F(ab′)2 fragment, or an Fvfragment. In another embodiment, an antibody is a humanized antibody. Inanother embodiment, an antibody is a human antibody. In someembodiments, an antibody comprises a framework having a human germlinesequence. In another embodiment, an antibody comprises a heavy chainconstant domain selected from the group consisting of IgG, IgG1, IgG2,IgG2A, IgG2B, IgG2C, IgG3, IgG4, IgA1, IgA2, IgD, IgM, and IgE constantdomains. In some embodiments, an antibody comprises a constant domain ofIgG4. In other embodiments, an antibody comprises a constant domain ofIgG4 having a backbone substitution of Ser to Pro that produces anIgG1-like hinge and permits formation of inter-chain disulfide bonds. Inanother embodiment, an antibody is conjugated to an agent selected fromthe group consisting of a fluorescent agent, a luminescent agent, anenzymatic agent and a radioactive agent.

In another embodiment, an antibody specifically binds topro/latent-Myostatin compared with mature myostatin. In someembodiments, an antibody specifically binds to pro/latent-Myostatincompared with another member of the transforming growth factor Betafamily. In another embodiment, said member is GDF11 or Activin.

A further aspect of the disclosure includes an antibody thatspecifically binds pro/latent-Myostatin and that inhibits proteolyticformation of mature myostatin by a tolloid protease. In someembodiments, said antibody inhibits proteolytic formation of maturemyostatin by a tolloid protease with an IC50 of less than 1 μM. In someembodiments, an antibody is cross-reactive with human and murinepro/latent-Myostatin. In other embodiments, the antibody specificallybinds to pro/latent-Myostatin compared with GDF11 or Activin. In anotherembodiment, an antibody specifically binds to pro/latent-Myostatincompared with mature myostatin.

Another aspect of the disclosure encompasses a method of reducingmyostatin receptor activation in cells present in a medium comprisingpro/latent-Myostatin, the method comprising delivering to the medium anantibody described above in an amount effective for inhibitingproteolytic activation of the pro/latent-Myostatin. In some embodiments,the medium further comprises a proprotein convertase. In otherembodiments, the medium further comprises a tolloid protease. In anotherembodiment, an antibody is delivered to the medium in an amounteffective for inhibiting proteolytic activation of thepro/latent-Myostatin by the tolloid protease. In some embodiments, thecell is in vitro. In other embodiments, the cell is in vivo.

Another aspect of the disclosure includes a method of treating a subjecthaving a myopathy, the method comprising administering to the subject aneffective amount of an antibody described above. In some embodiments,the myopathy is a primary myopathy. In another embodiment, the primarymyopathy comprises disuse atrophy. In other embodiments, the disuseatrophy is associated with hip fracture, elective joint replacement,critical care myopathy, spinal cord injury or stroke. In someembodiments, the myopathy is a secondary myopathy, in which muscle lossis secondary to a disease pathology. In other embodiments, the secondarymyopathy comprises denervation, genetic muscle weakness or cachexia. Inanother embodiment, the secondary myopathy is a denervation associatedwith amyotrophic lateral sclerosis or spinal muscular atrophy. In someembodiments, the secondary myopathy is a genetic muscle weaknessassociated with a muscular dystrophy. In other embodiments, thesecondary myopathy is a cachexia associated with renal failure, AIDS, acardiac condition, cancer or aging.

Another aspect of the disclosure includes a method of treating a subjecthaving a disease or condition related to aging. Exemplary diseases andconditions related to ageing include, without limitation, sarcopenia(age-related muscle loss), frailty, and androgen deficiency.

Another aspect of the disclosure includes a method of treating a subjecthaving a disease or condition related to disuse atrophy/trauma.Exemplary diseases and conditions related to disuse atrophy/traumainclude, without limitation, muscle weakness related to time spent in anintensive care unit (ICU), hip/joint replacement, hip fracture, stroke,bed rest, SCI, rotator cuff injury, knee replacement, bone fracture, andburns.

Another aspect of the disclosure includes a method of treating a subjecthaving a neurodegenerative disease or condition. Exemplaryneurodegenerative diseases or conditions include, without limitation,spinal muscular atrophy and amyotrophic lateral sclerosis (ALS).

Another aspect of the disclosure includes a method of treating a subjecthaving a disease or condition related to Cachexia. Exemplary diseasesand conditions related to cachexia include, without limitation, cancer,chronic heart failure, acquired immune deficiency syndrome (AIDS),chronic obstructive pulmonary disease (COPD), and chronic kidney disease(CKD).

Another aspect of the disclosure includes a method of treating a subjecthaving a disease or condition related to rare diseases. Exemplary rarediseases and conditions include, without limitation, osteogenesisimperfecta, sporadic Inclusion body myositis, and acute lymphoblasticleukemia.

Another aspect of the disclosure includes a method of treating a subjecthaving a disease or condition related to a metabolic disorder and/orbody composition. In some embodiments, the disease or condition isobesity (e.g., severe obesity), Prader-Willi, type II diabetes, oranorexia. However, additional diseases or conditions related tometabolic disorders and/or body composition are within the scope of thisdisclosure.

Another aspect of the disclosure includes a method of treating a subjecthaving a disease or condition related to congenital myopathies.Exemplary congenital myopathies include, without limitation, X-linkedmyotubular myopathy, autosomal dominant centronuclear myopathy,autosomal recessive centronuclear myopathy, nemaline myopathy, andcongenital fiber-type disproportion myopathy.

Another aspect of the disclosure includes a method of treating a subjecthaving a disease or condition related to muscular dystrophies. Exemplarymuscular dystrophies include, without limitation, Duchenne's, Becker's,facioscapulohumeral (FSH), and Limb-Girdle muscular dystrophies.

Another aspect of the disclosure includes a method of treating a subjecthaving a urogynecological related disease or condition, glotticdisorders (stenosis), extraocular myopathy, carpel tunnel,Guillain-Barré, or osteosarcoma.

In some embodiments, treatment results in improved muscle strength inthe subject. In other embodiments, treatment results in improvedmetabolic status in the subject.

In some embodiments, an antibody is administered at a dose in a range of0.1 mg/kg to 100 mg/kg. In another embodiment, an antibody isadministered at a dose in a range of 0.3 mg/kg to 30 mg/kg.

In some embodiments, an antibody is administered to the subjectintravenously. In other embodiments, an antibody is administered to thesubject subcutaneously. In another embodiment, an antibody isadministered to the subject on multiple occasions. In some embodiments,said multiple administrations are performed at least monthly. In anotherembodiment, said multiple administrations are performed at least weekly.

A further aspect of the disclosure includes a composition comprising anyantibody described above and a carrier. In some embodiments, saidcarrier is a pharmaceutically acceptable carrier. In other embodiments,an antibody and carrier are in a lyophilized form. In anotherembodiment, an antibody and carrier are in solution. In someembodiments, an antibody and carrier are frozen. In other embodiments,an antibody and carrier are frozen at a temperature less than or equalto −65° C.

Other aspects of the disclosure include an isolated nucleic acidencoding a protein comprising three complementarity determining regions(CDRs): CDRH1, CDRH2, and CDRH3, in which CDRH3 comprises a sequence asset forth in SEQ ID NO: 10 or 11. In some embodiments, said CDRH1comprises a sequence as set forth in SEQ ID NO: 1, 2 or 3.

In other embodiments, CDRH2 comprises a sequence as set forth in any oneof SEQ ID NOs: 4-9.

Another aspect of the present disclosure includes an isolated nucleicacid encoding a protein comprising three complementarity determiningregions (CDRs): CDRL1, CDRL2, and CDRL3, in which CDRL3 comprises asequence as set forth in SEQ ID NO: 22. In some embodiments, said CDRL1comprises a sequence as set forth in any one of SEQ ID NOs: 12-17. Inother embodiments, CDRL2 comprises a sequence as set forth in any one ofSEQ ID NOs: 18-21.

Further aspects of the present disclosure include an isolated nucleicacid comprising a sequence as set forth in any one of SEQ ID NOs: 38-49.

Another aspect of the disclosure includes an isolated cell comprising anisolated nucleic acid described above.

The present disclosure, in some aspects, includes methods of assessing abiological sample obtained from a subject having a myopathy. In someembodiments, the method comprises: preparing an immunological reactionmixture that comprises protein of a biological sample obtained from thesubject and an antibody that specifically binds pro/latent-Myostatin;maintaining the immunological reaction mixture under conditions thatpermit binding complexes to form between the antibody and apro/latent-Myostatin; and determining the extent of binding complexformation. In some embodiments, the method comprises: preparing animmunological reaction mixture that comprises protein of a biologicalsample obtained from the subject and an antibody that specifically bindspro-Myostatin; maintaining the immunological reaction mixture underconditions that permit binding complexes to form between the antibodyand a pro-Myostatin; and determining the extent of binding complexformation. In some embodiments, the method comprises: preparing animmunological reaction mixture that comprises protein of a biologicalsample obtained from the subject and an antibody that specifically bindslatent-Myostatin; maintaining the immunological reaction mixture underconditions that permit binding complexes to form between the antibodyand a latent-Myostatin; and determining the extent of binding complexformation. In some embodiments, the method comprises: preparing animmunological reaction mixture that comprises protein of a biologicalsample obtained from the subject and an antibody that specifically bindsmature-Myostatin; maintaining the immunological reaction mixture underconditions that permit binding complexes to form between the antibodyand a mature-Myostatin; and determining the extent of binding complexformation.

In one aspect, disclosed herein is an isolated antibody comprising aheavy chain variable region comprising an amino acid sequence of SEQ IDNO:25 and a light chain variable region comprising an amino acidsequence of SEQ ID NO:31. In one embodiment, the antibody comprises aheavy chain comprising an amino acid sequence of SEQ ID NO:50. Inanother embodiment, the antibody comprises a light chain comprising anamino acid sequence of SEQ ID NO:51.

In another aspect, disclosed herein is an isolated antibody comprising aheavy chain variable region comprising a CDRH1 sequence comprising SEQID NO:1, a CDRH2 sequence comprising SEQ ID NO:6, and a CDRH3 sequencecomprising SEQ ID NO:11; and a light chain variable region comprising aCDRL1 sequence comprising SEQ ID NO:14, a CDRL2 sequence comprising SEQID NO:20, and a CDRL3 sequence comprising SEQ ID NO:23.

In one embodiment, the heavy chain variable region comprises a sequenceof SEQ ID NO:26. In another embodiment, the light chain variable regioncomprises a sequence of SEQ ID NO:32.

In one embodiment, the heavy chain variable region comprises a sequenceof SEQ ID NO:27. In another embodiment, the light chain variable regioncomprises a sequence of SEQ ID NO:33

In another aspect, disclosed herein is an isolated antibody comprising aheavy chain variable region comprising a CDRH1 sequence comprising SEQID NO:1, a CDRH2 sequence comprising SEQ ID NO:8, and a CDRH3 sequencecomprising SEQ ID NO:11; and a light chain variable region comprising aCDRL1 sequence comprising SEQ ID NO:16, a CDRL2 sequence comprising SEQID NO:20, and a CDRL3 sequence comprising SEQ ID NO:23.

In one embodiment, the heavy chain variable region comprises a sequenceof SEQ ID NO:28. In another embodiment, the light chain variable regioncomprises a sequence of SEQ ID NO:34.

In one embodiment, the heavy chain variable region comprises a sequenceof SEQ ID NO:29. In one embodiment, the light chain variable regioncomprises a sequence of SEQ ID NO:35.

In one embodiment, the antibody is a human antibody. In one embodiment,the antibody comprises an IgG4 constant domain. In one embodiment, theantibody comprises an IgG4 constant domain having a backbonesubstitution of Ser to Pro that produces an IgG1-like hinge and permitsformation of inter-chain disulfide bonds.

In one embodiment, the antibody specifically binds topro/latent-myostatin. In one embodiment, the antibody specifically bindsto pro-myostatin. In another embodiment, the antibody specifically bindsto latent-myostatin. In one embodiment, the antibody does not bind tomature myostatin.

In one embodiment, the antibody inhibits proteolytic formation of maturemyostatin by tolloid protease. In one embodiment, the antibody inhibitsproteolytic formation of mature myostatin by tolloid protease with anIC50 of less than 1 μM.

In one embodiment, the antibody is cross-reactive with human and murinepro/latent-myostatin. In another embodiment, the antibody binds topro/latent-myostatin but does not bind to GDF11 or activin.

In one aspect, disclosed herein is a method of reducing myostatinreceptor activation in cells present in a medium comprisingpro/latent-myostatin, the method comprising delivering to the medium anantibody described herein in an amount effective for inhibitingproteolytic activation of the pro/latent-myostatin. In one embodiment,the medium comprises a proprotein convertase. In another embodiment, themedium comprises a tolloid protease. In one embodiment, the cell is invitro. In another embodiment, the cell is in vivo.

In another aspect, disclosed herein is a method of treating a subjecthaving a myopathy, the method comprising administering to the subject aneffective amount of an antibody disclosed herein.

In one embodiment, the myopathy is a primary myopathy. In anotherembodiment, the primary myopathy is disuse atrophy. In one embodiment,the disuse atrophy is associated with hip fracture, elective jointreplacement, critical care myopathy, spinal cord injury, and/or stroke.

In another embodiment, the myopathy is a secondary myopathy in whichmuscle loss is secondary to a disease pathology. In one embodiment, thesecondary myopathy comprises denervation, genetic muscle weakness, orcachexia. In another embodiment, the secondary myopathy is a denervationassociated with amyotrophic lateral sclerosis or spinal muscularatrophy. In yet another embodiment, the secondary myopathy is a geneticmuscle weakness associated with a muscular dystrophy. In one embodiment,the secondary myopathy is a cachexia associated with renal failure,AIDS, a cardiac condition, cancer, or aging.

In one embodiment, the administering results in improved muscle strengthin the subject. In one embodiment, the administering results in improvedmetabolic status in the subject.

In one embodiment, the antibody is administered at a dose in a range of0.1 mg/kg to 100 mg/kg. In another embodiment, the antibody isadministered at a dose in a range of 0.3 mg/kg to 30 mg/kg.

In one embodiment, the antibody is administered to the subjectintravenously. In another embodiment, the antibody is administered tothe subject subcutaneously.

In one embodiment, the antibody is administered to the subject onmultiple occasions. In one embodiment, the multiple administrations areperformed at least monthly. In another embodiment, the multipleadministrations are performed at least weekly.

In another aspect, disclosed herein is a pharmaceutical compositioncomprising an antibody disclosed herein and a pharmaceuticallyacceptable carrier. In one embodiment, the composition is a lyophilizedcomposition. In another embodiment, the composition is a liquidcomposition. In one embodiment, the composition is frozen. In oneembodiment, the composition is frozen at a temperature less than orequal to −65° C.

In another aspect, disclosed herein is a syringe comprising apharmaceutical composition described herein.

In another aspect, disclosed herein is an isolated nucleic acid encodingan antibody comprising a heavy chain variable region comprising anucleic acid sequence of SEQ ID NO:39 and a light chain variable regioncomprising a nucleic acid sequence of SEQ ID NO:45.

In another aspect, disclosed herein is an isolated nucleic acid encodingan antibody comprising a heavy chain variable region comprising a CDRH1sequence comprising SEQ ID NO:1, a CDRH2 sequence comprising SEQ IDNO:6, and a CDRH3 sequence comprising SEQ ID NO:11; and a light chainvariable region comprising a CDRL1 sequence comprising SEQ ID NO:14, aCDRL2 sequence comprising SEQ ID NO:20, and a CDRL3 sequence comprisingSEQ ID NO:23.

In one embodiment, the heavy chain variable region comprises a sequenceof SEQ ID NO:40. In one embodiment, the light chain variable regioncomprises a sequence of SEQ ID NO:46.

In one embodiment, the heavy chain variable region comprises a sequenceof SEQ ID NO:41. In another embodiment, the light chain variable regioncomprises a sequence of SEQ ID NO:47.

In another aspect, disclosed herein is an isolated nucleic acid encodingan antibody comprising a heavy chain variable region comprising a CDRH1sequence comprising SEQ ID NO:1, a CDRH2 sequence comprising SEQ IDNO:8, and a CDRH3 sequence comprising SEQ ID NO:11; and a light chainvariable region comprising a CDRL1 sequence comprising SEQ ID NO:16, aCDRL2 sequence comprising SEQ ID NO:20, and a CDRL3 sequence comprisingSEQ ID NO:23.

In one embodiment, the heavy chain variable region comprises a sequenceof SEQ ID NO:42. In another embodiment, the light chain variable regioncomprises a sequence of SEQ ID NO:48.

In one embodiment, the heavy chain variable region comprises a sequenceof SEQ ID NO:43. In another embodiment, the light chain variable regioncomprises a sequence of SEQ ID NO:49.

In another aspect, disclosed herein is an isolated cell comprising anisolated nucleic acid described herein.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B show Myostatin domain structure and proMyostatin assembly.FIG. 1A shows Myostatin secreted as a proprotein, with an inhibitoryprodomain followed by a C-terminal growth factor domain, which exists asa disulfide-linked dimer. FIG. 1B shows precursor protein assembled inan inactive conformation where the prodomain (dark gray) encloses thegrowth factor (light gray) with a “straightjacket” assembly. This figureis an adaption from the structure of latent TGFβ1 (Shi et al. Nature2011).

FIG. 2 shows the activation of Myostatin involves two distinct proteaseevents, generating three major Myostatin species. The biosyntheticprecursor protein, proMyostatin, is processed by two separate proteases.Cleavage of proMyostatin (and proGDF11) is carried out by a proproteinconvertase, such as Furin/PACE3 (Paired Basic Amino acid Cleaving Enzyme3) or PCSK5 (Proprotein Convertase Subtilisin/Kexin type 5), which cutsat a conserved RXXR site between the prodomain and mature growth factor.This cleavage produces a latent complex, in which the mature growthfactor is shielded from binding to its receptors by the prodomain.Activation and release of the active growth factor is accomplished aftercleavage by an additional protease from the BMP/tolloid family, such asTLL-2 (Tolloid-like protein 2) or BMP1 (Bone Morphogenetic Protein 1).These cleavage events yield a mature form of Myostatin, which may bereferred to as Active Myostatin or Mature Myostatin.

FIGS. 3A-3C show that Ab1 blocks cleavage of proMyostatin by members ofthe tolloid family of proteases. Latent Myostatin samples, preincubatedwith increasing amounts of Ab1, were analyzed in a myostatin activationassay. Following analysis of myostatin release by reporter assay (FIG.3A), samples were then run under reducing conditions and probed bywestern blot with an antibody raised towards the prodomain of Myostatin(FIG. 3B). An ˜18 kDa band (box), corresponding to the ARM portion ofthe prodomain generated after tolloid cleavage, decreased proportionallywith increasing doses of Ab1. The latent and proMyostatin standards (45ng loaded) show the migration of proMyostatin at ˜50 kDa, and theprodomain at ˜37 kDa. FIG. 3C shows the activation of Myostatin involvestwo distinct protease events, generating three major Myostatin species.The biosynthetic precursor protein, proMyostatin, is processed by twoseparate proteases. Cleavage of proMyostatin (and proGDF11) is carriedout by a proprotein convertase, such as Furin/PACE3 (Paired Basic Aminoacid Cleaving Enzyme 3) or PCSK5 (Proprotein Convertase Subtilisin/Kexintype 5), which cuts at a conserved RXXR site between the prodomain andmature growth factor. This cleavage produces a latent complex, in whichthe mature growth factor is shielded from binding to its receptors bythe prodomain. See FIG. 3B, which illustrates the potential inhibitionof a tolloid protease, blocking further cleavage of proMyostatin.Activation and release of the active growth factor is accomplished aftercleavage by an additional protease from the BMP/tolloid family, such asTLL-2 (Tolloid-like protein 2) or BMP1 (Bone Morphogenetic Protein 1).

FIG. 4 shows the performance of the parental Ab1 antibody and othercandidates in the cell-based reporter assay. Following an overnightproteolysis reaction with enzymes from both the proprotein-convertaseand tolloid protease families, the release of mature growth factor wasmeasured using a CAGA-based reporter assay in 293T cells. Results werecompared to control reactions to calculate the fraction of proMyostatinor proGDF11 which was released in the assay. Standard deviation for anaverage of 3 replicates is shown, but not visible on the graph for mostdata points due to their low magnitude.

FIG. 5 shows graphically that Ab1, Ab2, Ab4, and Ab6 antibodies do notinhibit proGDF11 activation.

FIG. 6 shows results of an assay evaluating mean percent body weightchange. Animals were weighed daily and the percent weight change fromDay 0 was calculated. Data represent group means±SEM. The mean percentchange data for each group on day 42 of the study were analyzed using aone-way ANOVA followed by Holm-Sidak's post hoc test in comparison tothe PBS Control Group, **p<0.01.

FIGS. 7A-7D show results of an assay evaluating tissue weights. FIG. 7Ashows the mean gastrocnemius weight. FIG. 7B shows the mean pectoralisweight. FIG. 7C shows the mean soleus weight. FIG. 7D shows the meantriceps weight. Statistical evaluation was performed using a one-wayANOVA followed by Holm-Sidak's post hoc test in comparison to theVehicle Control Group (Group 1). Data represent group means±SEM.**p<0.01. Bars indicate from left-to-right Groups 1-5.

FIGS. 8A-8C show results of an assay evaluating tissue weights. FIG. 8Ashows the mean tibialis anterior weight. FIG. 8B shows the meandiaphragm weight. FIG. 8C shows the mean quadriceps weight. Statisticalevaluation was performed using a one-way ANOVA followed by Holm-Sidak'spost hoc test in comparison to the Vehicle Control Group (Group 1). Datarepresent group means±SEM. *p<0.05. Bars indicate from left-to-rightGroups 1-5.

FIGS. 9A-9B show results of an assay evaluating mean percent body weightand lean mass change. FIG. 9A is a graph showing the calculated percentweight change from Day 0 in animals weighed twice weekly throughout thestudy. In FIG. 9B, animals underwent EchoMRI (QNMR) to measure bodycomposition on days −4, 7, 14, 21, and 28 and percent lean mass changefrom Day 0 was calculated. Data represent group means±SEM. For both bodyweight and lean mass the mean percent change data for each group on day28 of the study were analyzed using a one-way ANOVA followed byHolm-Sidak's post hoc test in comparison to the IgG Control Group (Group2). ***p<0.0005, **p<0.005, *p<0.05, ns (not significant).

FIGS. 10A-10D are graphs showing results of an assay evaluating muscleweights. FIG. 10A shows the mean quadriceps weight, FIG. 10B shows themean gastrocnemius weight, FIG. 10C shows the mean tibialis anteriorweight, and FIG. 10D shows the mean diaphragm weight. Percent differencein mean muscle weights of the Ab1 treated groups compared to the IgGcontrol group is noted above each bar. Statistical evaluation wasperformed using a one-way ANOVA followed by Holm-Sidak's post hoc testin comparison to the IgG Control Group (Group 2). Data represent groupmeans±SEM. ****p<0.0001, ***p<0.0005, **p<0.005, *p<0.05, ns (notsignificant).

FIGS. 11A-11B show results of an assay evaluating mean percent bodyweight and lean mass change. FIG. 11A shows the percent weight changefrom Day 0 calculated from animals weighed twice weekly throughout thestudy. (FIG. 11B) Animals underwent EchoMRI (QNMR) to measure bodycomposition on days −1, 6, and 13 and percent lean mass change from Day−1 was calculated. PBS=phosphate buffered saline, Dex=dexamethasone, IgG(20)=IgG control antibody dosed at 20 mg/kg/wk, Ab1 (20)=Ab1 antibodydosed at 20 mg/kg/wk, and Ab1 (2)=Ab1 antibody dosed at 2 mg/kg/wk. Datarepresent group means±SEM. Mean percent change data for each group onday 14 (for body weight) and day 13 (for lean mass) were analyzed usinga one-way ANOVA followed by a Dunnett's multiple comparisons test vs.group 1 (****p<0.0001, ***p<0.0005, **p<0.005, *p<0.05) and vs. group 5(++++p<0.0001, +++p<0.0005, ++p<0.005, +p<0.05). ns (not significant).

FIGS. 12A-12D are graphs showing results of an assay evaluating theweights of different muscles. FIG. 12A shows the mean gastrocnemiusweight (grams), FIG. 12B shows the mean quadriceps weight (grams), FIG.12C shows the mean percent gastrocnemius weight change versus thecontrol animals treated with PBS (IP) and normal drinking water (Group1), and FIG. 12D shows the mean percent quadriceps weight change versusthe control animals treated with PBS (IP) and normal drinking water(Group 1). PBS=phosphate buffered saline, Dex=dexamethasone, IgG(20)=IgG control antibody dosed at 20 mg/kg/wk, Ab1 (20)=Ab1 antibodydosed at 20 mg/kg/wk, and Ab1 (2)=Ab1 antibody dosed at 2 mg/kg/wk. ForFIGS. 12A-12B, error bars represent standard deviation (SD). For FIGS.12C-12D, error bars represent standard error of the mean (SEM).Statistical evaluation was performed using a one-way ANOVA followed by aDunnett's multiple comparisons test vs. group 1 (****p<0.0001,***p<0.0005, **p<0.005, *p<0.05) and vs. group 5 (++++p<0.0001,+++p<0.0005, ++p<0.005, +p<0.05). ns (not significant). Bars indicatefrom left-to-right, PBS, water; PBS, dex; IgG Control; Ab1(20); andAb1(2).

FIGS. 13A-13B show results of an assay evaluating the mean percent bodyweight and lean mass change. FIG. 13A shows the percent weight changefrom Day 0 calculated for animals who were weighed twice weeklythroughout the study. FIG. 13B shows the percent lean mass change fromDay −1 calculated from animals who underwent EchoMRI (QNMR) to measurebody composition on days −1, 7, and 14. PBS=phosphate buffered saline,IgG (20)=IgG control antibody dosed at 20 mg/kg/wk, Ab1 (20)=Ab1antibody dosed at 20 mg/kg/wk, and Ab1 (2)=Ab1 antibody dosed at 2mg/kg/wk. Data represent group means±SEM.

FIGS. 14A-14D show results of an assay evaluating muscle weights. FIG.14A shows the mean gastrocnemius weight from the casted leg (grams),FIG. 14B shows the mean quadriceps weight from the casted leg (grams),FIG. 14C shows the mean percent gastrocnemius weight change versus thecontrol animals treated with PBS (IP) and not casted (Group 1), and FIG.14D shows the mean percent quadriceps weight change versus the controlanimals treated with PBS (IP) and not casted (Group 1). PBS=phosphatebuffered saline, IgG (20)=IgG control antibody dosed at 20 mg/kg/wk, Ab1(20)=Ab1 antibody dosed at 20 mg/kg/wk, and Ab1 (2)=Ab1 antibody dosedat 2 mg/kg/wk. For FIGS. 14A-14B, error bars represent standarddeviation (SD). For FIGS. 14C-14D, error bars represent standard errorof the mean (SEM). Statistical evaluation was performed using a one-wayANOVA followed by a Dunnett's multiple comparisons test vs. group 1(****p<0.0001, ***p<0.0005, **p<0.005, *p<0.05) and vs. group 5(++++p<0.0001, +++p<0.0005, ++p<0.005, +p<0.05). ns (not significant).Bars indicate from left-to-right, PBS, no cast; PBS, casted; IgGControl(2), casted; Ab1(20), casted; and Ab1(2), casted.

FIG. 15 shows results of an assay evaluating the lean mass change at Day21 (top right) and Day 28 (top left). It also depicts the percent changein lean mass at three different doses, 20 mg/kg/wk (bottom left), 2mg/kg/wk (bottom middle), and 0.5 mg/kg/wk (bottom right) of the testedantibodies, PBS control, and IgG control. Statistical evaluation wasperformed using a one-way ANOVA followed by a Dunnett's multiplecomparisons test vs. group 1 (****p<0.0001, ***p<0.005, **p<0.01,*p<0.05) and vs. the IgG control. For the top two panels, bars fromleft-to-right are: PBS; IgG Ctrl 20 mg/kg/wk; Ab1 20 mg/kg/wk; Ab1 2mg/kg/wk; Ab1 0.5 mg/kg/wk; Ab2 20 mg/kg/wk; Ab2 2 mg/kg/wk; Ab2 0.5mg/kg/wk; Ab4 20 mg/kg/wk; Ab4 2 mg/kg/wk; Ab4 0.5 mg/kg/wk; Ab6 20mg/kg/wk; Ab6 2 mg/kg/wk; and Ab6 0.5 mg/kg/wk. For the bottom leftpanel (20 mg/kg/wk), the data points corresponding to day 28 postdosing, from top to bottom, correspond to Ab1, Ab4, Ab2, Ab6, IgGcontrol, and PBS. For the bottom center panel (2 mg/kg/wk), the datapoints corresponding to day 28 post dosing, from top to bottom,correspond to Ab2, Ab1, Ab6, Ab4, IgG control, and PBS. For the bottomright panel (0.5 mg/kg/wk), the data points, corresponding to day 28post dosing, from top to bottom, correspond to IgG control Ab1, Ab2,PBS, Ab4, and Ab6.

FIGS. 16A-16B show the domain structure and evaluation of Myostatinprecursor forms. FIG. 16A shows the domain structure of proMyostatin andlatent Myostatin, with protease cleavage sites indicated. FIG. 16B showspartially proprotein convertase cleaved proMyostatin run on an SDS PAGEgel. Under reducing conditions, the protein bands consisted of theproMyostatin monomer (˜50 kD), prodomain (˜37 kD) and growth factor(12.5 kD).

FIGS. 17A-17B show Ab1 is specific for Myostatin. FIG. 17A shows Ab1binds specifically to proMyostatin and latent Myostatin, with no bindingobserved to other members of the TGFB superfamily, most notably thecorresponding forms of GDF11. Ab1 was administered at a highconcentration (50 ug/mL) to Forte-Bio BLI tips coated with the indicatedantigen and the on and off rates were measured to obtain an approximateKd value. The magnitude of biosensor response, indicating a bindingevent, is graphically represented by black bars, and the calculated Kdis indicated in orange. FIG. 17B shows that Ab1 blocks the activation ofproMyostatin, but not proGDF11. Following an overnight proteolysisreaction with enzymes from both the proprotein-convertase and tolloidprotease families, the release of mature growth factor was measuredusing a CAGA-based reporter assay in 293T cells. Results were comparedto control reactions to calculate the fraction of proMyostatin orproGDF11 which was released in the assay.

FIGS. 18A-18C show the SCID dose response with the candidate antibodies.FIG. 18A shows the muscle weight of the gastrocnemius and FIG. 18B showsthe muscle weight of the quadriceps. FIG. 18C shows the percent changesin mean muscle weight compared to the PBS control. The bars in FIGS.18A-18B from left-to-right are: PBS; IgG Ctrl 20 mg/kg/wk; Ab1 20mg/kg/wk; Ab1 2 mg/kg/wk; Ab1 0.5 mg/kg/wk; Ab2 20 mg/kg/wk; Ab2 2mg/kg/wk; Ab2 0.5 mg/kg/wk; Ab4 20 mg/kg/wk; Ab4 2 mg/kg/wk; Ab4 0.5mg/kg/wk; Ab6 20 mg/kg/wk; Ab6 2 mg/kg/wk; and Ab6 0.5 mg/kg/wk.

FIG. 19 shows the results of a duration of action study comparing Ab1 toan existing myostatin antibody (AbMyo). PBS was used as a negativecontrol; IgG was used as a positive control. The lean mas change wasexamined under different dosing protocols after 21 days.

FIG. 20 is a schematic illustrating an assay that reconstitutesMyostatin activation in vitro.

FIGS. 21A-21B show the heavy chain (FIG. 21A; SEQ ID NO: 50) and lightchain (FIG. 21B; SEQ ID NO: 51) of a humanized monoclonal antibody (Ab2)of the IgG4 subtype with Proline substituted for Serine. This generatesan IgG1-like hinge sequence and minimizes the incomplete formation ofinter-chain disulfide bridges which is characteristic of IgG4. Thecomplementarity-determining regions (CDRs) are underlined. Bolded NSTsequence: N-linked glycosylation consensus sequence site; Bolded DPsequences are potential cleavage sites; Bolded NX sequences, wherein Xcan be S, T, or G are potential deamidation sites; Bolded DX sequences,wherein X can be G, S, T, or SDG are potential isomerization sites;Bolded methionines are potential methionine oxidation sites; Bolded Q isan expected N-terminal pyroglutamic acid.

FIG. 22 is a schematic showing the reduced immunogenicity risk bygermlining. 24H4 (WT) contains 5 non-germline amino acids withinframework regions, as indicated in the schematic.

FIGS. 23A-23C show the optimization of Ab1. Optimized candidates whichbind specifically to proMyostatin were chosen, resulting in dozens ofclones with increased affinity. FACS was performed to show the increasedbinding of the yeast clones (FIG. 23B) compared to Ab1 (FIG. 23A). FIG.23C shows the affinity-matured variants have a slower off-rate by octetas well.

FIGS. 24A-24B show sequence alignments of the variable heavy regions(FIG. 24A) and variable light regions (FIG. 24B) of parental Ab1 withaffinity optimized variants, Ab3 and Ab5. Sequence identifiers from topto bottom correspond to SEQ ID NOs.: 24, 26, 28 (FIG. 24A). Sequenceidentifiers from top to bottom correspond to SEQ ID NOs.: 30, 32, 34(FIG. 24B). Complementarity-determining regions (CDRs) are defined usingthe Kabat (underlined) and IMGT nomenclature (bold). Substitutions fromparental Ab1 are shown in light gray.

FIG. 25 shows the expression of pro- and latent-Myostatin in muscle andplasma from normal and atrophic mice.

FIG. 26 shows the quantitation of changes in pro and latent Myostatin inmuscle and plasma. The bars from left to right show proMyostatin, latentmyostatin, proMyostatin, latent myostatin, and latent myostatin.

FIG. 27 shows that Ab2 uniquely recognizes proMyostatin and latentMyostatin, binding to the major forms of Myostatin in both serum andmuscle. Non-reducing Western blot for prodomain (darker gray) and themature growth factor (lighter gray). Recombinant proMyostatin(rProMyostatin) shows the migration of proMyostatin and myostatinprodomain (latent myostatin) on the gel, highlighted by arrows. Inserum, both Ab2 and AbMyo bind to latent Myostatin (prodomain band) andmultiple partially processed precursors, however only Ab2 recognizedproMyostatin (top band). In muscle, Ab2 precipitated proMyostatin, withno interaction of AbMyo with proMyostatin in the muscle tissue.

FIGS. 28A-28B provide a model for Myostatin flux in normal and atrophicmuscle. In normal muscle (FIG. 28A), proMyostatin is produced in muscleand converted to latent Myostatin through cleavage by Furin protease,which may occur either inside or outside of the cell. Some fraction ofthe latent Myostatin in muscle is then released into the circulation,forming a circulating pool of latent Myostatin. In muscle atrophy (FIG.28B), an increase in the active Myostatin growth factor is caused byupregulation of proMyostatin levels in muscle and increased conversionof latent Myostatin to the active growth factor. As a consequence,circulating latent Myostatin is decreased as the muscle pool of latentMyostatin is redirected towards formation of mature Myostatin by mTLL2cleavage.

FIG. 29 shows detection of Ab2 (top line) and IgG control (bottom line)antibody in serum from dosed rats. Ab2 exhibits elevated levels in thecirculation as compared to the IgG control, with an average of 17.1μg/ml of Ab2 in serum at the end of the study. Ab2 levels determined byhuman IgG-specific ELISA with known quantities of each antibody used asa reference standard.

FIGS. 30A-30B shows pharmacodynamic effects of Ab2 in treated rats. FIG.30A shows rats treated with Ab2 show increased lean mass compared toPBS- or IgG control-treated animals. Ab2 and IgG were administeredintravenously at 10 mg/kg doses on day 0. Lean mass measured by qNMR(N=8 per group) at 7, 14, 21 and 28 days after dosing. FIG. 30B showsrectus femoris and tibilais anterior muscles were collected from allgroups at the end of the study (N=8 per group) and weighed to determinemuscle mass. Rats treated with Ab2 show an increase of 14% and 11% inrectus femoris and tibialis anterior muscle masses, respectively.

FIGS. 31A-31B shows pro/latent-Myostatin pro/latent-Myostatin levels inAb2-treated rats. FIG. 31A shows treatment with Ab2 (top line) increaseslatent myostatin levels in rat serum by ˜20-fold. FIG. 31B shows that inrat muscle (rectus femoris), Ab2 treatment leads to a 1.9× increase inthe latent form of Myostatin. The bars from left to right correspond toproMyostatin, latent Myostatin, proMyostatin, and latent Myostatin. Nostatistically significant change in proMyostatin is observed in ratmuscle. These data are from quantitative western analyses with n=3samples per group.

FIG. 32 shows treatment with Ab2 (Ab2) or with the comparator antibody(AbMyo) leads to increased lean mass by as early 7 days after antibodydosing. Increases in lean mass are equivalent for Ab2 and AbMyo until 21days after dosing. By 28 days after dosing, however, increases in leanmass are lost in the AbMyo-treated group, while increases in theAb2-treated group are maintained throughout the duration of the study.The top line corresponds to Ab2, the middle line correspond to AbMyo,and the bottom line corresponds to IgG Control (5 mg/kg).

FIG. 33 shows that after a single, 5 mg/kg dose of Ab2 (top line) or ofcomparator antibody (AbMyo; bottom line), serum levels of drug weremeasured using an anti-human IgG ELISA. Drug is detected in serum asearly as 1 hour after dosing, and levels >1 μg/ml of both antibodies canbe detected throughout the study. However, Ab2 exhibits a significantlylonger half-life and inferred area under the curve (AUCINF) than AbMyo,suggesting that, at similar doses, Ab2 exhibits significantly greaterexposure than AbMyo.

FIG. 34 shows serum Myostatin was measured in drug treated mice and incontrols using fluorescent western blotting. Despite the increased serumexposure of Ab2, serum latent Myostatin levels in both Ab2- andAbMyo-treated mice were similar. These data suggest that the circulatinglevels of free drug are sufficiently in excess to the level of targetthat the increased serum exposure of Ab2 does not lead to a greaterincrease in circulating latent Myostatin than is observed in the AbMyogroup. Groups of data from left to right correspond to IgG, Ab2, AbMyo,IgG, Ab2, and AbMyo.

FIGS. 35A-35B shows relative levels of latent and proMyostatin weremeasured in mouse muscle lysates by fluorescent western blot. FIG. 35Ashows latent Myostatin is elevated in both Ab2- and AbMyo-treatedmuscles. However, elevation of latent Myostatin in AbMyo-treated musclesreturns to baseline by day 28, while those in Ab2 treated muscles remainelevated until at least this time (P<0.003 vs. AbMyo treatment). FIG.35B shows a similar trend is observed with proMyostatin, though thedifference between the Ab2 and AbMyo treated groups at day 28 is notstatistically significant (P=0.068).

FIG. 36A shows effects of treatment with Ab2 on muscle mass and functionin mice.

FIG. 36B shows effects of treatment with Ab2 on maximal force generationin mice.

DETAILED DESCRIPTION

Myostatin is a member of the TGFβ superfamily, and belongs to asubfamily including two members: Myostatin (also known as GDF8) andGDF11. Like other members of the TGFβ superfamily, Myostatin and GDF11are both initially expressed as inactive precursor polypeptides (termedproMyostatin and proGDF11, respectively). The domain structure andnomenclature are shown in FIG. 1A. FIG. 1B illustrates a cartoon modelof the overall structure of proMyostatin, where the mature growth factoris held locked in a cage comprised of two alpha helices connected by aloop termed the “latency lasso”.

Activation and release of the mature growth factor is accomplished byseveral discrete protease cleavage events, outlined in FIG. 2. The firstcleavage step of proMyostatin and proGDF11 is carried out by aproprotein convertase, which cuts at a conserved RXXR site between theprodomain and mature growth factor. This cleavage produces a latentcomplex, in which the mature growth factor is shielded from binding toits receptors by the prodomain. Activation and release of the mature,active Myostatin growth factor is accomplished after cleavage by anadditional protease from the BMP/tolloid family, such as mTLL-2 (FIG.2).

Exemplary proGDF8 sequences in the human, rat, mouse and cynomolgus areprovided below. In these proGDF8 sequences, a proprotein convertasecleavage site is indicated in bold and a tolloid protease site isindicated by underlining. In some embodiments, the proprotein convertasecleavage site comprises amino acid residues 240 to 243 of SEQ ID NOs:52-55. In some embodiments, the tolloid protease site comprises aminoacid residues 74-75 of SEQ ID NOs: 52-55. It should be appreciated thatthe exemplary proGDF8 sequences provided herein are not intended to belimiting and additional proGDF8 sequences from other species, includingany isoforms thereof, are within the scope of this disclosure.

proGDF8 (human): (SEQ ID NO: 52)NENSEQKENVEKEGLCNACTWRQNTKSSRIEAIKIQILSKLRLETAPNISKDVIRQLLPKAPPLRELIDQYDVQRDDSSDGSLEDDDYHATTETIITMPTESDFLMQVDGKPKCCFFKFSSKIQYNKVVKAQLWIYLRPVETPTTVFVQILRLIKPMKDGTRYTGIRSLKLDMNPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKVTDTPKRSRRDFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLQKYPHTHLVHQANPRGSAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMVVDRCGCS. proGDF8 (rat):(SEQ ID NO: 53) NEDSEREANVEKEGLCNACAWRQNTRYSRIEAIKIQILSKLRLETAPNISKDAIRQLLPRAPPLRELIDQYDVQRDDSSDGSLEDDDYHATTETIITMPTESDFLMQADGKPKCCFFKFSSKIQYNKVVKAQLWIYLRAVKTPTTVFVQILRLIKPMKDGTRYTGIRSLKLDMSPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKVTDTPKRSRRDFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLQKYPHTHLVHQANPRGSAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMVVDRCGCS. proGDF8 (mouse):(SEQ ID NO: 54) NEGSEREENVEKEGLCNACAWRQNTRYSRIEAIKIQILSKLRLETAPNISKDAIRQLLPRAPPLRELIDQYDVQRDDSSDGSLEDDDYHATTETIITMPTESDFLMQADGKPKCCFFKFSSKIQYNKVVKAQLWIYLRPVKTPTTVFVQILRLIKPMKDGTRYTGIRSLKLDMSPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKVTDTPKRSRRDFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLQKYPHTHLVHQANPRGSAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMVVDRCGCS. proGDF8 (cynonnolgus):(SEQ ID NO: 55) NENSEQKENVEKEGLCNACTWRQNTKSSRIEAIKIQILSKLRLETAPNISKDAIRQLLPKAPPLRELIDQYDVQRDDSSDGSLEDDDYHATTETIITMPTESDFLMQVDGKPKCCFFKFSSKIQYNKVVKAQLWIYLRPVETPTTVFVQILRLIKPMKDGTRYTGIRSLKLDMNPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKVTDTPKRSRRDFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIA.

Myostatin and GDF11 share a relatively high degree of conservationbetween their mature growth factor domains, with ninety percentidentity, but are much less well conserved in their prodomain regionswith less than fifty percent amino acid identity between the two.Myostatin and GDF11 bind and signal through the same receptorsconsisting of a Type I receptor (ALK4/5) in association with a type IIreceptor (ACTRIIA/B). Engagement of Myostatin with Type I and Type IIreceptors initiates a signaling cascade leading to SMAD phosphorylationand transcriptional activation of muscle atrophy genes. The relativelyhigh degree of conservation in the mature growth factors has made itchallenging to identify reagents, such as monoclonal antibodies, thatcan differentiate between mature Myostatin and GDF11.

In some embodiments, pro/latent-Myostatin antibodies are provided hereinthat specifically bind to a chimeric construct that contains the growthfactor domain and N terminal propeptide portion of GDF11 and the Cterminal portion of the propeptide of GDF8. This chimeric construct, asforth below, is referred as GDF11Arm8.

>GDF11 Arm8 (SEQ ID NO: 65)MDMRVPAQLLGLLLLWFSGVLGDYKDDDDKHHHHHHLEVLFQGPAEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAPEPDGCPVCVWRQHSRELRLESIKSQILSKLRLKEAPNISREVVKQLLPKAPPLRELIDQYDVQRDDSSDGSLEDDDYHATTETIITMPTESDFLMQVDGKPKCCFFKFSSKIQYNKVVKAQLWIYLRPVETPTTVFVQILRLIKPMKDGTRYTGIRSLKLDMNPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKVTDTPKRSRRNLGLDCDEHSSESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGQCEYMFMQKYPHTHLVQQANPRGSAGPCCTPTKMSPINMLYFNDKQQIIYGKIP GMVVDRCGCS

Role of Myostatin in Myopathies

Skeletal muscle accounts for approximately 40% of body mass and is adynamic organ, turning over at a rate of 1-2% per day. Muscle atrophy isa highly regulated catabolic process which occurs during periods ofdisuse (e.g. disuse atrophy) and/or in response to heightened systemicinflammation (cachexia). In disuse atrophy, which can occur duringprolonged periods of immobilization such as during bed rest, muscle lossoccurs rapidly. For example, during a hospital stay of one week, anaverage patient loses ˜1.3 kg of muscle mass.

Muscle atrophy causes significant morbidity in a wide range of clinicalconditions. In diseases of denervation like amyotrophic lateralsclerosis (ALS) or spinal muscular atrophy (SMA) and genetic diseasesincluding muscular dystrophies, loss of muscle strength and function arehighly disabling clinical manifestations for which there are no adequatetreatments. In cachexia syndromes due to renal failure, AIDS, cardiacconditions, or cancer, muscle wasting often undermines successfultreatment of the primary condition. Muscle loss also results as anatural process of aging and in its most severe form is categorized assarcopenia, a pervasive condition among the elderly that is increasinglybeing recognized as a pathology warranting intervention. Lastly, a majordriver of muscle atrophy is disuse. Immobilization causes rapid andsignificant muscle loss in a large group of conditions such as hipfracture, elective joint replacement, spinal cord injury, critical caremyopathy and stroke. While varied in their cause, these indicationsshare a characteristic of muscle weakness, which leads to significantdisability, lengthy physical rehabilitation and recovery times andimpairment of quality of life.

There has been an unmet medical need in muscle atrophy conditions.Accordingly, in some embodiments, methods are provided herein fortreating muscle atrophy. In some embodiments, methods provided hereinrelate to the treatment of a primary myopathy. In some embodiments,methods provided herein relate to the treatment of secondary myopathy,such as, for example, diseases of denervation, genetic muscle weaknessand cachexia, conditions in which muscle loss is secondary to thedisease pathology. In some embodiments, methods provided herein for thetreatment of primary myopathies, such as disuse atrophy (e.g.,associated with hip fracture or spinal cord injury (SCI)), result inincrease in muscle mass, strength and function in a subject.

Myostatin Pathway Inhibition

There are several Myostatin pathway antagonists in various stages ofclinical development towards the treatment of muscle-related conditions.Such pathway antagonists target either the mature growth factor or itstype II receptor, and most antagonize the signaling of multiple TGFβfamily members. For example, a number of current clinical candidatesblock additional growth factors such as Activin A, GDF11, and BMPs 9 and10, which are regulators of reproductive biology, wound healing,erythropoiesis and blood vessel formation, respectively. Aspects of thisdisclosure relate to a recognition that blocking these factors inaddition to Myostatin will potentially limit the population of patientswho can safely undergo therapy due to unacceptable side-effects.

Accordingly, provided herein are antibodies capable of binding toproMyostatin and/or latent Myostatin, thereby inhibiting Myostatinactivity, and uses thereof for treating diseases and disordersassociated with myopathy. In some embodiments, given the prevalence ofthe latent complex in circulation, treatments are provided herein thatspecifically target more abundant and longer-lived Myostatin precursorse.g., proMyostatin and latent Myostatin, rather than the mature growthfactor. Without wishing to be bound by any particular theory, antibodiesprovided herein may prevent the proteolytic activation of proMyostatinand/or latent Myostatin into mature Myostatin which is considered the“active” form of Myostatin, capable of activating the Myostatin pathway,e.g., by binding Type I (ALK4/5) and Type II (ACTRIIA/B) receptors.

As used herein, the term “pro/latent-Myostatin” refers to proMyostatin,latent Myostatin, or both. In some embodiments, ananti-pro/latent-Myostatin antibody binds specifically to proMyostatin.In some embodiments, an anti-pro/latent-Myostatin antibody bindsspecifically to latent Myostatin. In some embodiments, ananti-pro/latent-Myostatin antibody binds specifically to both latentMyostatin and proMyostatin. It should be appreciated that “latentMyostatin” and “proMyostatin” may also be referred to herein as “latentGDF8” and “proGDF8”, respectively.

As used herein, the term “mature myostatin” refers to a mature,biologically active form of myostatin. In some embodiments, maturemyostatin is capable of myostatin receptor binding and/or activation.Activation and release of mature myostatin in vivo from itspro-myostatin form is accomplished by several discrete protease cleavageevents. To begin with, “pro-myostatin” is cleaved by a proproteinconvertase, resulting in “latent-myostatin,” in which the maturemyostatin is shielded from binding to its receptors by a portion of theprodomain. Activation and release of mature myostatin is accomplishedafter cleavage of latent-myostatin by an additional protease from theBMP/tolloid family, such as mTLL-2. See, for example, FIGS. 1A, 1B, and2. As used herein, the term “mature myostatin” can refer to bothfull-length mature myostatin, as well as fragments of the full-lengthmature myostatin which retain biological activity. Exemplary maturemyostatin sequences, variants thereof, and methods of generating maturemyostatin are well known in the art and described in more detail herein.

The term “pro-myostatin,” also known as “proGDF8,” refers to an inactiveprecursor of mature myostatin which comprises a disulfide-linkedhomodimer, each molecule of the homodimer comprising the amino terminalprodomain covalently bound to the carboxyl terminal mature myostatindomain. In one embodiment, “pro-myostatin” has not been cleaved byeither a proprotein convertase, or a protease from the BMP/tolloidfamily. Exemplary pro-myostatin sequences, variants thereof, and methodsof generating pro-myostatin are well known in the art and described inmore detail herein.

As used herein the term “latent-myostatin” refers to an inactiveprecursor of mature myostatin which comprises a disulfide-linkedhomodimer, each molecule of the homodimer comprising the amino terminalprodomain non-covalently bound to the carboxyl terminal mature myostatindomain. In one embodiment, “latent-myostatin” is generated from apro-myostatin that has been cleaved by a proprotein convertase, butwhich has not been cleaved by a protease from the BMP/tolloid family. Inanother embodiment, “latent-myostatin” can be generated by combining theprodomain and the carboxy terminal mature myostatin domain in vitro andallowing them to fold properly. See, for example, Sengle et al., J.Biol. Chem., 286(7):5087-5099, 2011. Exemplary latent-myostatinsequences, variants thereof, and methods of generating latent-myostatinare well known in the art and described in more detail herein.

As used herein, the term “pro/latent-myostatin” refers to pro-myostatin,latent-myostatin, or both pro-myostatin and latent-myostatin. In oneembodiment, an antibody disclosed herein binds to pro-myostatin. Inanother embodiment, an antibody disclosed herein binds tolatent-myostatin. In another embodiment, an antibody disclosed hereinbinds to pro-myostatin and latent-myostatin.

As used herein, the term “pure pro-myostatin” or “pure pro-GDF8” refersto a composition comprising pro-myostatin that is free, or substantiallyfree, of other forms of myostatin, such as latent-myostatin and maturemyostatin. In one embodiment, an antibody disclosed herein specificallybinds pure pro-myostatin. In other words, such an antibody binds topro-myostatin in a composition which lacks the other forms ofmyostation, latent-myostatin and mature myostatin.

As used herein, the term “proprotein convertase cleavage site” refers toa site where pro-myostatin is cleaved by a proprotein convertase. In oneembodiment, a proprotein convertase cleavage site is a conserved RXXRsite between the prodomain and the biologically active domain, or maturemyostatin. See, for example, FIGS. 1A, 1B, and 2.

As used herein, the term “BMP/tolloid protease family cleavage site”refers to a site where latent myostatin is cleaved by a BMP/tolloidprotease family member. In one embodiment, a BMP/tolloid protease familymember is mTLL-2. See, for example, FIGS. 1A, 1B, and 2.

Antibodies that Bind Pro/Latent-Myostatin

The present disclosure is based, at least in part, on the surprisingdiscovery that certain pro/latent-Myostatin-specific antibodies (e.g.,an antibody referred to herein as Ab1), prevented proteolytic activationof pro/latent-Myostatin into mature Myostatin. Furthermore, inhibitionof Myostatin activation using such antibodies was effective forincreasing muscle mass in both dexamethasone and casting induced muscleatrophy mouse models. Aspects of the disclosure provide antibodies(e.g., antibodies and antigen binding fragments) that bind topro/latent-Myostatin and inhibit proteolytic activation ofpro/latent-Myostatin into mature Myostatin.

An antibody (interchangeably used in plural form) is an immunoglobulinmolecule capable of specific binding to a target, such as acarbohydrate, polynucleotide, lipid, polypeptide, etc., through at leastone antigen recognition site, located in the variable region of theimmunoglobulin molecule. As used herein, the term “antibody” encompassesnot only intact (e.g., full-length) polyclonal or monoclonal antibodies,but also antigen-binding fragments thereof (such as Fab, Fab′, F(ab′)2,Fv), single chain (scFv), mutants thereof, fusion proteins comprising anantibody portion, humanized antibodies, chimeric antibodies, diabodies,linear antibodies, single chain antibodies, multispecific antibodies(e.g., bispecific antibodies) and any other modified configuration ofthe immunoglobulin molecule that comprises an antigen recognition siteof the required specificity, including glycosylation variants ofantibodies, amino acid sequence variants of antibodies, and covalentlymodified antibodies. An antibody includes an antibody of any class, suchas IgD, IgE, IgG, IgA, or IgM (or sub-class thereof), and the antibodyneed not be of any particular class. Depending on the antibody aminoacid sequence of the constant domain of its heavy chains,immunoglobulins can be assigned to different classes. There are fivemajor classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, andseveral of these may be further divided into subclasses (isotypes),e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy-chain constantdomains that correspond to the different classes of immunoglobulins arecalled alpha, delta, epsilon, gamma, and mu, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds pro/latent-myostatin is substantially free of antibodies thatspecifically bind antigens other than pro/latent-myostatin). An isolatedantibody that specifically binds pro/latent-myostatin may, however, havecross-reactivity to other antigens, such as pro/latent-myostatinmolecules from other species. Moreover, an isolated antibody may besubstantially free of other cellular material and/or chemicals.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences and fragments thereof. The humanantibodies of the disclosure may include amino acid residues not encodedby human germline immunoglobulin sequences (e.g., mutations introducedby random or site-specific mutagenesis in vitro or by somatic mutationin vivo), for example in the CDRs and in particular CDR3. However, theterm “human antibody”, as used herein, is not intended to includeantibodies in which CDR sequences derived from the germline of anothermammalian species, such as a mouse, have been grafted onto humanframework sequences.

The term “epitope” includes any polypeptide determinant capable ofspecific binding to an immunoglobulin or T-cell receptor. In certainembodiments, epitope determinants include chemically active surfacegroupings of molecules such as amino acids, sugar side chains,phosphoryl, or sulfonyl, and, in certain embodiments, may have specificthree dimensional structural characteristics, and/or specific chargecharacteristics. An epitope is a region of an antigen that is bound byan antibody. In certain embodiments, an antibody is said to specificallybind an antigen when it preferentially recognizes its target antigen ina complex mixture of proteins and/or macromolecules.

Antibodies described herein are capable of binding to apro/latent-Myostatin, thereby inhibiting the proteolytic activation ofpro/latent-Myostatin into mature Myostatin. In some instances,antibodies described herein can inhibit the proteolytic activation ofpro/latent-Myostatin by at least 20%, e.g., 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, or higher. In some instances, antibodies described hereincan inhibit the proteolytic cleavage of proMyostatin by a proproteinconvertase (e.g., furin) by at least 20%, e.g., 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, or higher. In some instances antibodies described hereincan inhibit the proteolytic cleavage of proMyostatin or latent Myostatinby a tolloid protease (e.g., mTLL2) by at least 20%, e.g., 30%, 40%,50%, 60%, 70%, 80%, 90%, 95%, or higher. The inhibitory activity of ananti-pro/latent-Myostatin antibody can be measured by routine methods,for example, by Western blot analysis as described in Example 1 and FIG.3. However, it should be appreciated that additional methods may be usedfor measuring the inhibitory activity of an anti-pro/latent-Myostatinantibody on proteolytic cleavage of pro/latent-Myostatin. In someembodiments, inhibition of pro/latent-Myostatin cleavage (e.g., by aproprotein convertase and/or tolloid protease) may be reflected as aninhibition constant (Ki), which provides a measure of inhibitor potency,and which it is the concentration of inhibitor (e.g., ananti-pro/latent-Myostatin antibody) required to reduce protease activity(e.g., of a proprotein convertase or tolloid protease) by half and isnot dependent on enzyme or substrate concentrations.

In some embodiments, a proprotein convertase comprises (i) a catalyticdomain that hydrolyzes a peptide bond of a protein containing aproprotein convertase cleavage site, and (ii) a binding pocket thatbinds to an rTGF with a proprotein convertase cleavage site. Examples ofproprotein convertases for use in accordance with the present disclosureinclude, without limitation, PCSK5/6, PACE4, PACE7 and PACE3 (e.g.,furin). A proprotein convertase, in some embodiments, is obtained fromany mammal including, without limitation, humans, monkeys or rodents(e.g., mice, rats, hamsters).

In some embodiments, a proprotein convertase is homologous to aproprotein convertase selected from the group consisting of: PCSK5/6,PACE4, PACE7 and PACE3 (e.g., furin). For example a proproteinconvertase may be at least 70% identical, at least 80% identical, atleast 90% identical, at least 95% identical, at least 96% identical, atleast 97% identical, at least 98% identical, at least 99% identical, atleast 99.5% identical, or at least about 99.9% identical to PCSK5/6,PACE4, PACE7 or PACE3 (e.g., furin).

A proprotein convertase cleavage site, in some embodiments, is an aminosequence that can be cleaved by a proprotein convertase (e.g., PCSK5/6,PACE4, PACE7 and PACE3). In some embodiments, the proprotein convertasecleavage site comprises the amino acid sequence R-X-X-R, where R isarginine and X is any amino acid. In some embodiments, the proproteinconvertase cleavage site comprises the amino acid sequence R-X-(K/R)-R,where R is arginine, K is lysine and X is any amino acid. In someembodiments, the proprotein convertase cleavage site comprises the aminoacid sequence is R-V-R-R (SEQ ID NO: 57), where R is arginine and V isvaline. Exemplary proprotein convertase cleavage sites for human, rat,mouse, and cynomolgus myostatin are shown, in bold, in SEQ ID NOs:52-55. In some embodiments, the proprotein convertase cleavage sitecomprises the amino acid sequence RSRR (SEQ ID NO: 56).

In some embodiments, tolloid proteases for use in accordance with thepresent disclosure include, without limitation, BMP-1, mTLL-1 andmTLL-2. A tolloid protease may be obtained from any mammal including,without limitation, humans, monkeys, or rodents (e.g., mice, rats,hamsters). In some embodiments, a tolloid protease is homologous to atolloid protease selected from the group consisting of: BMP-1, mTLL-1and mTLL-2. For example a tolloid protease may be at least 70%identical, at least 80% identical, at least 90% identical, at least 95%identical, at least 96% identical, at least 97% identical, at least 98%identical, at least 99% identical, at least 99.5% identical, or at leastabout 99.9% identical to BMP-1, mTLL-1 and mTLL-2.

A tolloid protease cleavage site, in some embodiments, is an aminosequence that can be cleaved by a tolloid (e.g., BMP-1, mTLL-1 andmTLL-2). Exemplary tolloid protease cleavage sites for human, rat,mouse, and cynomolgus myostatin are shown, in underlining, in SEQ IDNOs: 52-55. In some embodiments, the tolloid cleavage site comprises theamino acid sequence QR, where Q is glutamine and R is arginine.

In some embodiments, antibodies described herein are capable of bindingto a pro/latent-Myostatin, thereby inhibiting Myostatin activity. Insome instances, the antibodies described herein can inhibit Myostatinsignaling by at least 20%, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,or higher. In some embodiments, inhibition of Myostatin signaling can bemeasured by routine methods, for example, using a Myostatin activationassay as described in Example 1. However, it should be appreciated thatadditional methods may be used for measuring Myostatin signalingactivity.

It should be appreciated that the extent of proteolytic cleavage ofmyostatin, e.g., by a proprotein convertase and/or a tolloid protease,can be measured and/or quantified using any suitable method. In someembodiments, the extent of proteolytic cleavage of myostatin is measuredand/or quantified using an enzyme-linked immunosorbent assay (ELISA).For example, an ELISA may be used to measure the level of releasedgrowth factor (e.g., mature myostatin). As another example, an antibodythat specifically binds to proMyostatin, latent Myostatin and/or matureMyostatin can be used in an ELISA to measure the level of a specificform of myostatin (e.g., pro/latent/mature-Myostatin), to quantify theextent of proteolytic cleavage of myostatin. In some embodiments, theextent of proteolytic cleavage of myostatin is measured and/orquantified using immunoprecipitation followed by SDS-PAGE or massspectrometry of tryptic peptides, fluorescence anisotropy-basedtechniques, FRET assays, hydrogen-deuterium-exchange mass spectrometry,and/or NMR spectroscopy.

In some embodiments, antibodies, also known as immunoglobulins, aretetrameric glycosylated proteins composed of two light (L) chains ofapproximately 25 kDa each and two heavy (H) chains of approximately 50kDa each. Two types of light chain, termed lambda and kappa, may befound in antibodies. Depending on the amino acid sequence of theconstant domain of heavy chains, immunoglobulins can be assigned to fivemajor classes: A, D, E, G, and M, and several of these may be furtherdivided into subclasses (isotypes), e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁,and IgA₂. Each light chain typically includes an N-terminal variable (V)domain (V_(L)) and a constant (C) domain (C_(L)). Each heavy chaintypically includes an N-terminal V domain (V_(H)), three or four Cdomains (C_(H)1-3), and a hinge region. The C_(H) domain most proximalto V_(H) is designated as C_(H)1. The V_(H) and V_(L) domains consist offour regions of relatively conserved sequences called framework regions(FR1, FR2, FR3, and FR4), which form a scaffold for three regions ofhypervariable sequences (complementarity determining regions, CDRs). TheCDRs contain most of the residues responsible for specific interactionsof the antibody with the antigen. CDRs are referred to as CDR1, CDR2,and CDR3. Accordingly, CDR constituents on the heavy chain are referredto as CDRH1, CDRH2, and CDRH3, while CDR constituents on the light chainare referred to as CDRL1, CDRL2, and CDRL3. The CDRs typically refer tothe Kabat CDRs, as described in Sequences of Proteins of ImmunologicalInterest, US Department of Health and Human Services (1991), eds. Kabatet al. Another standard for characterizing the antigen binding site isto refer to the hypervariable loops as described by Chothia. See, e.g.,Chothia, D. et al. (1992) J. Mol. Biol. 227:799-817; and Tomlinson etal. (1995) EMBO J. 14:4628-4638. Still another standard is the AbMdefinition used by Oxford Molecular's AbM antibody modeling software.See, generally, e.g., Protein Sequence and Structure Analysis ofAntibody Variable Domains. In: Antibody Engineering Lab Manual (Ed.:Duebel, S, and Kontermann, R., Springer-Verlag, Heidelberg). Embodimentsdescribed with respect to Kabat CDRs can alternatively be implementedusing similar described relationships with respect to Chothiahypervariable loops or to the AbM-defined loops, or combinations of anyof these methods.

In some embodiments, anti-pro/latent-Myostatin antibodies of the presentdisclosure and the nucleic acid molecules of the present disclosure thatencode the antibodies include the CDR amino acid sequences shown inTable 1.

TABLE 1 CDRH1 CDRH2 CDRH3 CDRL1 CDRL2 CDRL3 (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID (SEQ ID Antibody NOs: 1-3) NOs: 4-9) NOs: 10-11)NOs: 12-17) NOs: 18-21) NOs: 22-23) Ab1 Kabat: SSYGMH (SEQ VISYDGSNKYYDLLVRFLEWSH SGSSSNIGSNTV SDNQRPS (SEQ AAWDDSLNGV IMGT: ID NO: 1)ADSVKG (SEQ YYGMDV (SEQ H (SEQ ID NO: ID NO: 18) (SEQ ID NO: GFTFSSYGMHID NO: 4) ID NO: 10) 12) SDN (SEQ ID 22) (SEQ ID NO: ISYDGSN (SEQSSNIGSNT (SEQ NO: 19) 2) ID NO: 5) ID NO: 13) Ab3 Kabat: SSYGMH (SEQVISYDGSIKYYA DLLVRFLEWSH SGSTSNIGSNTV SDDQRPS (SEQ AAWDESLNGV IMGT:ID NO: 1) DSVKG (SEQ ID KYGMDV (SEQ H (SEQ ID NO: ID NO: 20) (SEQ ID NO:GFAFSSYGMH NO: 6) ID NO: 11) 14) SDD (SEQ ID 23) (SEQ ID NO:ISYDGSI (SEQ TSNIGSNT (SEQ NO: 21) 3) ID NO: 7) ID NO: 15) Ab5 Kabat:SSYGMH (SEQ VISYDGNNKYY DLLVRFLEWSH SGSSSNIGGNTV SDDQRPS (SEQ AAWDESLNGVIMGT: ID NO: 1) ADSVKG (SEQ KYGMDV (SEQ H (SEQ ID NO: ID NO: 20)(SEQ ID NO: GFAFSSYGMH ID NO: 8) ID NO: 11) 16) SDD (SEQ ID 23)(SEQ ID NO: ISYDGNN (SEQ SSNIGGNT (SEQ NO: 21) 3) ID NO: 9) ID NO: 17)

In Table 1, the single sequences of CDRH3 and CDRL3 reflect Kabat andIMGT.

In some embodiments, anti-pro/latent-Myostatin binding agents (e.g.,antibodies) of the disclosure include any antibody (including antigenbinding fragments) that includes a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, orCDRL3, or combinations thereof, as provided for any one of theantibodies shown in Table 1. In some embodiments,anti-pro/latent-Myostatin binding agents include the CDRH1, CDRH2,CDRH3, CDRL1, CDRL2, and CDRL3 of any one of the antibodies shown inTable 1. The disclosure also includes any nucleic acid sequence thatencodes a molecule comprising a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, orCDRL3 as provided for any one of the antibodies shown in Table 1.Antibody heavy and light chain CDR3 domains may play a particularlyimportant role in the binding specificity/affinity of an antibody for anantigen. Accordingly, the anti-pro/latent-Myostatin binding agents ofthe disclosure, or the nucleic acid molecules thereof, may include atleast the heavy and/or light chain CDR3s of antibodies as shown in Table1.

Aspects of the disclosure relate to a monoclonal antibody or antigenbinding fragment, that binds to pro/latent-Myostatin protein and thatcomprises six complementarity determining regions (CDRs): CDRH1, CDRH2,CDRH3, CDRL1, CDRL2, and CDRL3.

In some embodiments, CDRH1 comprises a sequence as set forth in any oneof SEQ ID NOs: 1-3. In some embodiments, CDRH2 comprises a sequence asset forth in any one of SEQ ID NOs: 4-9. In some embodiments, CDRH3comprises a sequence as set forth in any one of SEQ ID NOs: 10-11. CDRL1comprises a sequence as set forth in any one of SEQ ID NOs: 12-17. Insome embodiments, CDRL2 comprises a sequence as set forth in any one ofSEQ ID NOs: 18-21. In some embodiments, CDRL3 comprises a sequence asset forth in any one of SEQ ID NOs: 22-23.

In some embodiments (e.g., as for anti-pro/latent-Myostatin antibodyAb1, shown in Table 1), CDRH1 comprises a sequence as set forth in SEQID NO: 1 or 2, CDRH2 comprises a sequence as set forth in SEQ ID NO: 4or 5, CDRH3 comprises a sequence as set forth in SEQ ID NO: 10, CDRL1comprises a sequence as set forth in SEQ ID NO: 12, or 13, CDRL2comprises a sequence as set forth in SEQ ID NO: 18 or 19, and CDRL3comprises a sequence as set forth in SEQ ID NO: 22, and the antibodybinds to pro/latent-Myostatin.

In some embodiments (e.g., as for anti-pro/latent-Myostatin antibodyAb3, shown in Table 1), CDRH1 comprises a sequence as set forth in SEQID NO: 1 or 3, CDRH2 comprises a sequence as set forth in SEQ ID NO: 6or 7, CDRH3 comprises a sequence as set forth in SEQ ID NO: 11, CDRL1comprises a sequence as set forth in SEQ ID NO: 14, or 15, CDRL2comprises a sequence as set forth in SEQ ID NO: 20 or 21, and CDRL3comprises a sequence as set forth in SEQ ID NO: 23, and the antibodybinds to pro/latent-Myostatin.

In some embodiments (e.g., as for anti-pro/latent-Myostatin antibodyAb5, shown in Table 1), CDRH1 comprises a sequence as set forth in SEQID NO: 1 or 3, CDRH2 comprises a sequence as set forth in SEQ ID NO: 8or 9, CDRH3 comprises a sequence as set forth in SEQ ID NO: 11, CDRL1comprises a sequence as set forth in SEQ ID NO: 16, or 17, CDRL2comprises a sequence as set forth in SEQ ID NO: 20 or 21, and CDRL3comprises a sequence as set forth in SEQ ID NO: 23, and the antibodybinds to pro/latent-Myostatin. In some examples, any of theanti-pro/latent-Myostatin binding agents (e.g., antibodies) of thedisclosure include any antibody (including antigen binding fragments)having one or more CDR (e.g., CDRH or CDRL) sequences substantiallysimilar to CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and/or CDRL3. For example,the antibodies may include one or more CDR sequences as shown in Table 1(SEQ ID NOs: 1-23) containing up to 5, 4, 3, 2, or 1 amino acid residuevariations as compared to the corresponding CDR region in any one of SEQID NOs: 1-23. The complete amino acid and nucleic acid sequences for theheavy chain variable region and light chain variable region of theantibodies listed in Table 1 are provided below.

Heavy chain variable region - Ab1 parental (SEQ ID NO: 24)QIQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLLVRFLEWSHYYGMDVWGQGTTVTVSS (SEQ ID NO: 38)CAGATCCAGCTGGTGCAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATCTCCTGGTGCGATTTTTGGAGTGGTCGCACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA Heavy chain variable region - Ab2 germline(SEQ ID NO: 25) QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLLVRFLEWSHYYGMDVWGQGTTVTVSS (SEQ ID NO: 39)CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATCTCCTGGTGCGATTTTTGGAGTGGTCGCACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA Heavy chain variable region - Ab3 parental(SEQ ID NO: 26) QIQLVQSGGGVVQPGRSLRLSCAASGFAFSSYGMHWVRQAPGKGLEWVAVISYDGSIKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLLVRFLEWSHKYGMDVWGQGTTVTVSS (SEQ ID NO: 40)CAGATCCAGCTGGTGCAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCGCCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTATCAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATCTCCTGGTGCGATTTTTGGAGTGGTCGCACAAGTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA Heavy chain variable region - Ab4 germline(SEQ ID NO: 27) QVQLVESGGGVVQPGRSLRLSCAASGFAFSSYGMHWVRQAPGKGLEWVAVISYDGSIKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLLVRFLEWSHKYGMDVWGQGTTVTVSS (SEQ ID NO: 41)CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCGCCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTATCAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATCTCCTGGTGCGATTTTTGGAGTGGTCGCACAAGTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA Heavy chain variable region - Ab5 parental(SEQ ID NO: 28) QIQLVQSGGGVVQPGRSLRLSCAASGFAFSSYGMHWVRQAPGKGLEWVAVISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLLVRFLEWSHKYGMDVWGQGTTVTVSS (SEQ ID NO: 42)CAGATCCAGCTGGTGCAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCGCCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAATAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATCTCCTGGTGCGATTTTTGGAGTGGTCGCACAAGTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA Heavy chain variable region - Ab6 germline(SEQ ID NO: 29) QVQLVESGGGVVQPGRSLRLSCAASGFAFSSYGMHWVRQAPGKGLEWVAVISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLLVRFLEWSHKYGMDVWGQGTTVTVSS (SEQ ID NO: 43)CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCGCCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAATAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATCTCCTGGTGCGATTTTTGGAGTGGTCGCACAAGTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA Light chain variable region - Ab1 parental(SEQ ID NO: 30) QPVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVHWYQQLPGTAPKLLIYSDNQRPSGVPDRFSGSKSGTSASLVISGLQSDDEADYYCAAWDDSLNGVFGG GTKLTVL(SEQ ID NO: 44) CAGCCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCAGCTCCAACATCGGAAGTAATACTGTCCACTGGTACCAGCAACTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGTGATAATCAGCGCCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGTCATCAGTGGGCTCCAGTCTGACGATGAGGCTGATTATTACTGTGCAGCATGGGATGACAGCCTGAATGGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA Light chain variable region - Ab2 germline(SEQ ID NO: 31) QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVHWYQQLPGTAPKLLIYSDNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGVFGG GTKLTVL(SEQ ID NO: 45) CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCAGCTCCAACATCGGAAGTAATACTGTCCACTGGTACCAGCAACTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGTGATAATCAGCGCCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCAGTCTGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGACAGCCTGAATGGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA Light chain variable region - Ab3 parental(SEQ ID NO: 32) QPVLTQPPSASGTPGQRVTISCSGSTSNIGSNTVHWYQQLPGTAPKLLIYSDDQRPSGVPDRFSGSKSGTSASLVISGLQSDDEADYYCAAWDESLNGVFGG GTKLTVL(SEQ ID NO: 46) CAGCCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCACCTCCAACATCGGAAGTAATACTGTCCACTGGTACCAGCAACTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGTGATGATCAGCGCCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGTCATCAGTGGGCTCCAGTCTGACGATGAGGCTGATTATTACTGTGCAGCATGGGATGAGAGCCTGAATGGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA Light chain variable region - Ab4 germline(SEQ ID NO: 33) QSVLTQPPSASGTPGQRVTISCSGSTSNIGSNTVHWYQQLPGTAPKLLIYSDDQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDESLNGVFGG GTKLTVL(SEQ ID NO: 47) CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCACCTCCAACATCGGAAGTAATACTGTCCACTGGTACCAGCAACTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGTGATGATCAGCGCCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCAGTCTGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGAGAGCCTGAATGGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA Light chain variable region - Ab5 parental(SEQ ID NO: 34) QPVLTQPPSASGTPGQRVTISCSGSSSNIGGNTVHWYQQLPGTAPKLLIYSDDQRPSGVPDRFSGSKSGTSASLVISGLQSDDEADYYCAAWDESLNGVFGG GTKLTVL(SEQ ID NO: 48) CAGCCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCAGCTCCAACATCGGAGGAAATACTGTCCACTGGTACCAGCAACTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGTGATGATCAGCGCCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGTCATCAGTGGGCTCCAGTCTGACGATGAGGCTGATTATTACTGTGCAGCATGGGATGAGAGCCTGAATGGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA Light chain variable region - Ab6 germline(SEQ ID NO: 35) QSVLTQPPSASGTPGQRVTISCSGSSSNIGGNTVHWYQQLPGTAPKLLIYSDDQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDESLNGVFGG GTKLTVL(SEQ ID NO: 49) CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCAGCTCCAACATCGGAGGAAATACTGTCCACTGGTACCAGCAACTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGTGATGATCAGCGCCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCAGTCTGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGAGAGCCTGAATGGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA Ab2-Heavy Chain (SEQ ID NO: 50)QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLLVRFLEWSHYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG Ab2-Light Chain(SEQ ID NO: 51) QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVHWYQQLPGTAPKLLIYSDNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGST VEKTVAPTECS

In some embodiments, anti-pro/latent-Myostatin antibodies of thedisclosure include any antibody that includes a heavy chain variabledomain of any one of SEQ ID NOs: 24-29 or a light chain variable domainof any one of SEQ ID NOs: 30-35. In some embodiments,anti-pro/latent-Myostatin antibodies of the disclosure include anyantibody that includes the heavy chain variable and light chain variablepairs of SEQ ID NOs: 24 and 30; 25 and 31; 26 and 32; 27 and 33; 28 and34; or 29 and 35).

Aspects of the disclosure provide anti-pro/latent-Myostatin antibodieshaving a heavy chain variable and/or a light chain variable amino acidsequence homologous to any of those described herein. In someembodiments, the anti-pro/latent-Myostatin antibody comprises a heavychain variable sequence or a light chain variable sequence that is atleast 75% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to the heavychain variable sequence of any of SEQ ID NOs: 24-29 or a light chainvariable sequence of any one of SEQ ID NOs: 30-35. In some embodiments,the homologous heavy chain variable and/or a light chain variable aminoacid sequences do not vary within any of the CDR sequences providedherein. For example, in some embodiments, the degree of sequencevariation (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) may occur withina heavy chain variable and/or a light chain variable sequence excludingany of the CDR sequences provided herein.

The “percent identity” of two amino acid sequences is determined usingthe algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad.Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into theNBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol.Biol. 215:403-10, 1990. BLAST protein searches can be performed with theXBLAST program, score=50, word length=3 to obtain amino acid sequenceshomologous to the protein molecules of interest. Where gaps existbetween two sequences, Gapped BLAST can be utilized as described inAltschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. Whenutilizing BLAST and Gapped BLAST programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used.

In some embodiments, conservative mutations can be introduced into theCDRs or framework sequences at positions where the residues are notlikely to be involved in interacting with pro/latent-Myostatin asdetermined based on the crystal structure. As used herein, a“conservative amino acid substitution” refers to an amino acidsubstitution that does not alter the relative charge or sizecharacteristics of the protein in which the amino acid substitution ismade. Variants can be prepared according to methods for alteringpolypeptide sequence known to one of ordinary skill in the art such asare found in references which compile such methods, e.g. MolecularCloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, orCurrent Protocols in Molecular Biology, F. M. Ausubel, et al., eds.,John Wiley & Sons, Inc., New York. Conservative substitutions of aminoacids include substitutions made amongst amino acids within thefollowing groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G;(e) S, T; (f) Q, N; and (g) E, D.

In some embodiments, the antibodies provided herein comprise mutationsthat confer desirable properties to the antibodies. For example, toavoid potential complications due to Fab-arm exchange, which is known tooccur with native IgG4 mAbs, the antibodies provided herein may comprisea stabilizing ‘Adair’ mutation (Angal S., et al., “A single amino acidsubstitution abolishes the heterogeneity of chimeric mouse/human (IgG4)antibody,” Mol Immunol 30, 105-108; 1993), where serine 228 (EUnumbering; residue 241 Kabat numbering) is converted to prolineresulting in an IgG1-like (CPPCP (SEQ ID NO: 58)) hinge sequence.Accordingly, any of the antibodies may include a stabilizing ‘Adair’mutation or the amino acid sequence CPPCP (SEQ ID NO: 58).

Anti-pro/latent-Myostatin binding agents of this disclosure mayoptionally comprise antibody constant regions or parts thereof. Forexample, a V_(L) domain may be attached at its C-terminal end to a lightchain constant domain like Cκ or Cλ. Similarly, a V_(H) domain orportion thereof may be attached to all or part of a heavy chain likeIgA, IgD, IgE, IgG, and IgM, and any isotype subclass. Antibodies mayinclude suitable constant regions (see, for example, Kabat et al.,Sequences of Proteins of Immunological Interest, No. 91-3242, NationalInstitutes of Health Publications, Bethesda, Md. (1991)). Therefore,antibodies within the scope of this may disclosure include V_(H) andV_(L) domains, or an antigen binding portion thereof, combined with anysuitable constant regions.

In certain embodiments, the V_(H) and/or V_(L) domains may be revertedto germline sequence, e.g., the FR of these domains are mutated usingconventional molecular biology techniques to match those produced by thegermline cells. For example, the V_(H) and/or V_(L) domains may bereverted to germline sequence of IgHV3-30 (SEQ ID NO: 36) and/orIgLV1-44 (SEQ ID NO: 37), respectively. It should be appreciated thatany of the V_(H) and/or V_(L) domains may be reverted to any suitablegermline sequence. In other embodiments, the FR sequences remaindiverged from the consensus germline sequences.

IgHV3-30 (SEQ ID NO: 36)QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR IgLV1-44 (SEQ ID NO: 37)QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNG

In some embodiments, anti-pro/latent-Myostatin antibodies or antigenbinding fragments may or may not include the framework region of theantibodies shown in SEQ ID NOs: 24-35. In some embodiments,anti-pro-latent-Myostatin antibodies are murine antibodies and includemurine framework region sequences.

In some embodiments, an anti-pro/latent-Myostatin antibodies of thedisclosure can bind to pro/latent-Myostatin with relatively highaffinity, e.g., with a Kd less than 10⁻⁶ M, 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M,10⁻¹⁰ M, 10⁻¹¹ M or lower. For example, anti-pro/latent-Myostatinantibodies can bind to pro/latent-Myostatin with an affinity between 5pM and 500 nM, e.g., between 50 pM and 100 nM, e.g., between 500 pM and50 nM. The disclosure also includes antibodies or antigen bindingfragments that compete with any of the antibodies described herein forbinding to pro/latent-Myostatin and that have an affinity of 50 nM orlower (e.g., 20 nM or lower, 10 nM or lower, 500 pM or lower, 50 pM orlower, or 5 pM or lower). The affinity and binding kinetics of theanti-pro/latent-Myostatin antibody can be tested using any suitablemethod including but not limited to biosensor technology (e.g., OCTET orBIACORE).

An antibody that “specifically binds” to a target antigen, binds to thetarget antigen with greater affinity, avidity, more readily, and/or withgreater duration than it binds to non-target antigens. In someembodiments, antibodies are disclosed herein that specifically bindspro/latent-Myostatin. in some embodiments any of the antibodies providedherein bind at or near a tolloid cleavage site or at or near a tolloiddocking site of pro/latent-Myostatin. In some embodiments, an antibodybinds near a tolloid cleavage site or near a tolloid docking site if itbinds within 15 or fewer amino acid residues of the tolloid cleavagesite or tolloid docking site. In some embodiments, any of the antibodiesprovided herein bind within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14 or 15 amino acid residues of a tolloid cleavage site or tolloiddocking site. In some embodiments, an antibody binds at or near atolloid cleavage site of GDF8. For example, an antibody may bind anamino acid sequence as set forth in SEQ ID NO: 62PKAPPLRELIDQYDVQRDDSSDGSLEDDDYHAT (SEQ ID NO: 62). In other embodiments,any of the antibodies provided herein bind at or near a proproteinconvertase cleavage site or at or near a proprotein convertase dockingsite of pro/latent-Myostatin. In some embodiments, an antibody bindsnear a proprotein convertase cleavage site or near a proproteinconvertase docking site if it binds within 15 or fewer amino acidresidues of the proprotein convertase cleavage site or proproteinconvertase docking site. In some embodiments, any of the antibodiesprovided herein bind within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14 or 15 amino acid residues of a proprotein convertase cleavage site orproprotein convertase docking site. In some embodiments, an antibodybinds at or near a proprotein convertase cleavage site of GDF8. Forexample, an antibody may bind an amino acid sequence as set forth in SEQID NO: 63.

(SEQ ID NO: 63) GLNPFLEVKVTDTPKRSRRDFGLDCDEHSTESRC.

In one example, the anti-pro/latent-Myostatin antibodies describedherein specifically bind pro/latent-Myostatin as compared to other formsof Myostatin and/or other members of the TGFβ family of growth factors.Members of the TGFβ family of growth factors include, without limitationAMH, ARTN, BMP10, BMP15, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8A,BMP8B, GDF1, GDF10, GDF11, GDF15, GDF2, GDF3, GDF3A, GDF5, GDF6, GDF7,GDF8, GDF9, GDNF, INHA, INHBA, INHBB, INHBC, INHBE, LEFTY1, LEFTY2,NODAL, NRTN, PSPN, TGFβ1, TGFβ2, and TGFβ3 protein. Such antibodies maybind pro/latent-Myostatin at a much higher affinity as compared to othermembers of the TGFβ family of growth factors (e.g., at least 2-fold,5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, or 1,000-foldhigher). In some embodiments, such antibodies may bindpro/latent-Myostatin with an affinity of at least 1,000 greater ascompared to other members of the TGFβ family of growth factors. In someembodiments, antibodies provided herein may bind to pro/latent-Myostatinat a much higher affinity as compared to one or more forms of GDF11 ormature Myostatin (e.g., at least 2-fold, 5-fold, 10-fold, 50-fold,100-fold, 200-fold, 500-fold, or 1,000-fold higher). In someembodiments, antibodies provided herein may bind to pro/latent-Myostatinwith an affinity of at least 1,000 greater as compared to one or moreforms of GDF11 (e.g., proGDF11, latent GDF11 or mature GDF11) or matureMyostatin Alternatively, or in addition, antibodies may exhibit a muchhigher inhibitory activity against proteolytic cleavage ofpro/latent-Myostatin (e.g., by a proprotein convertase or tolloidprotease) as compared with other members of the TGFβ family, such aspro/latent GDF11 (e.g., at least 2-fold, 5-fold, 10-fold, 50-fold,100-fold, 200-fold, 500-fold, 1,000-fold higher).

In some embodiments, antibodies bind an antigen but cannot effectivelyeliminate the antigen from the plasma. Thus, in some embodiments, theconcentration of the antigen in the plasma may be increased by reducingthe clearance of the antigen. However, in some embodiments, antibodies(e.g., sweeping antibodies) provided herein have an affinity to anantigen that is sensitive to pH. Such pH sensitive antibodies may bindto the antigen in plasma at neutral pH and dissociate from the antigenin an acidic endosome, thus reducing antibody-mediated antigenaccumulation and/or promoting antigen clearance from the plasma.

Aspects of the disclosure relate to sweeping antibodies. As used herein“sweeping antibodies” refer to antibodies having both pH-sensitiveantigen binding and at least a threshold level of binding to cellsurface neonatal Fc receptor (FcRn) at neutral or physiological pH. Insome embodiments, sweeping antibodies bind to the neonatal Fc receptorFcRn at neutral pH. For example sweeping antibodies may bind to the FcRnat a pH ranging from 7.0 to 7.6. In some embodiments, sweepingantibodies can bind to an antigen at an antigen binding site and bind toa cellular FcRn via an Fc portion of the antibody. In some embodiments,sweeping antibodies may then be internalized, releasing antigen in anacidic endosome, which may be degraded. In some embodiments, a sweepingantibody, no longer bound to the antigen, may then be released (e.g., byexocytosis) by the cell back into the serum.

In some embodiments, FcRn in the vascular endothelia (e.g., of asubject) extends the half-life of a sweeping antibody. In someembodiments, vascular endothelial cells internalize sweeping antibodies,which in some embodiments are bound to an antigen such as Myostatin(e.g., proMyostatin, latent Myostatin or primed Myostatin). In someembodiments, a sweeping antibody is recycled back into the bloodstream.In some embodiments, a sweeping antibody has an increased half-life(e.g., in the serum of a subject) as compared to its conventionalcounterpart. In some embodiments, a conventional counterpart of asweeping antibody refers the antibody from which the sweeping antibodywas derived (e.g., prior to engineering the Fc portion of theconventional antibody to bind FcRn with greater affinity at pH 7). Insome embodiments, a sweeping antibody has a half-life in the serum of asubject that is at least 1%, 5%, 10%, 15%, 20%, 25%, 35%, 50%, 75%,100%, 150%, 200% or 250% longer as compared to its conventionalcounterpart.

In some embodiments, an Fc portion of a sweeping antibody binds FcRn. Insome embodiments, the Fc portion of a sweeping antibody binds to FcRn ata pH of 7.4 with a Kd ranging from 10⁻³ M to 10⁻⁸ M. In someembodiments, a sweeping antibody binds to FcRn at a pH of 7.4 with a Kdranging from 10⁻³ M to 10⁻⁷ M, from 10⁻³ M to 10⁻⁶ M, from 10⁻³ M to10⁻⁵ M, from 10⁻³ M to 10⁻⁴ M, from 10⁻⁴ M to 10⁻⁸ M, from 10⁻⁴ M to10⁻⁷ M, from 10⁻⁴ M to 10⁻⁶ M, from 10⁻⁴ M to 10⁻⁵ M, from 10⁻⁵ M to10⁻⁸ M, from 10⁻⁵ M to 10⁻⁷ M, from 10⁻⁵ M to 10⁻⁶ M, from 10⁻⁶ M to10⁻⁸ M, from 10⁻⁶ M to 10⁻⁷ M, or from 10⁻⁷ M to 10⁻⁸ M. In someembodiments, FcRn binds to the CH2-CH3 hinge region of a sweepingantibody. In some embodiments, FcRn binds to the same region as proteinAor protein G. In some embodiments, FcRn binds to a different bindingsite from FcγRs. In some embodiments, the amino acid residues AA of asweeping antibody Fc region are required for binding to FcRn. In someembodiments, the amino acid residues AA of a sweeping antibody Fc regionaffect binding to FcRn.

In some embodiments, any of the antibodies provided herein areengineered to bind FcRn with greater affinity. In some embodiments, anyof the antibodies provided herein are engineered to bind FcRn withgreater affinity at pH 7.4. In some embodiments, the affinity ofsweeping antibodies to FcRn is increased to extend their pharmacokinetic(PK) properties as compared to their conventional counterparts. Forexample, in some embodiments, sweeping antibodies elicit less adversereactions due to their efficacy at lower doses. In some embodiments,sweeping antibodies are administered less frequently. In someembodiments, transcytosis of sweeping antibodies to certain tissue typesare increased. In some embodiments, sweeping antibodies enhanceefficiency of trans-placental delivery. In some embodiments, sweepingantibodies are less costly to produce.

In some embodiments, any of the antibodies provided herein areengineered to bind FcRn with lower affinity. In some embodiments, any ofthe antibodies provided herein are engineered to bind FcRn with loweraffinity at pH 7.4. In some embodiments, the affinity of sweepingantibodies to FcRn is decreased to shorten their pharmacokinetic (PK)properties as compared to their conventional counterparts. For example,in some embodiments, sweeping antibodies are more rapidly cleared forimaging and/or radioimmunotherapy. In some embodiments, sweepingantibodies promote clearance of endogenous pathogenic antibodies as atreatment for autoimmune diseases. In some embodiments, sweepingantibodies reduce the risk of adverse pregnancy outcome, which may becaused by trans-placental transport of material fetus-specificantibodies.

In some embodiments, sweeping antibodies have decreased affinity to anantigen at low pH as compared to a neutral or physiological pH (e.g., pH7.4). In some embodiments, sweeping antibodies have a decreased affinityto an antigen at an acidic pH (e.g. a pH ranging from 5.5 to 6.5) ascompared to a physiological pH (e.g., pH 7.4). It should be appreciatedthat any of the antibodies provided herein can be engineered todissociate from the antigen depending on changes in pH (e.g., pHsensitive antibodies). In some embodiments, sweeping antibodies providedherein are engineered to bind antigen dependent on pH. In someembodiments, sweeping antibodies provided herein are engineered to bindFcRn dependent on pH. In some embodiments, sweeping antibodies providedherein are internalized by endocytosis. In some embodiments, sweepingantibodies provided here are internalized by FcRn binding. In someembodiments, endocytosed sweeping antibodies release antigen in anendosome. In some embodiments, sweeping antibodies are recycled back tothe cell surface. In some embodiments, sweeping antibodies remainattached to cells. In some embodiments, endocytosed sweeping antibodiesare recycled back to the plasma. It should be appreciated that the Fcportion of any of the antibodies provided herein may be engineered tohave different FcRn binding activity. In some embodiments, FcRn bindingactivity affects the clearance time of an antigen by a sweepingantibody. In some embodiments, sweeping antibodies may be long-acting orrapid-acting sweeping antibodies.

In some embodiments, converting a conventional therapeutic antibody intoa sweeping antibody reduces the efficacious dose. In some embodiments,converting a conventional therapeutic antibody into a sweeping antibodyreduces the efficacious dose by at least 1%, 2%, 5%, 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 95%, or 99%. In some embodiments, converting aconventional therapeutic antibody into a sweeping antibody reduces theefficacious dose by at least 1.5 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6fold, 8 fold, 10 fold, 15 fold, 20 fold, 50 fold or 100 fold.

In some embodiments, selecting an appropriate dose of a sweepingantibody for therapy may be performed empirically. In some embodiments,a high dose of a sweeping antibody may saturate FcRn, resulting inantibodies which stabilize antigen in serum without being internalized.In some embodiments, a low dose of a sweeping antibody may not betherapeutically effective. In some embodiments, sweeping antibodies areadministered once a day, once a week, once every two weeks, once everythree weeks, once every four weeks, once every 6 weeks, once every 8weeks, once every 10 weeks, once every 12 weeks, once every 16 weeks,once every 20 weeks, or once every 24 weeks.

In some embodiments, any of the antibodies provided herein may bemodified or engineered to be sweeping antibodies. In some embodiments,any of the antibodies provided herein may be converted into a sweepingantibody using any suitable method. For example, suitable methods formaking sweeping antibodies have been previously described in Igawa etal., (2013) “Engineered Monoclonal Antibody with Novel Antigen-SweepingActivity In Vivo,” PLoS ONE 8(5): e63236; and Igawa et al.,“pH-dependent antigen-binding antibodies as a novel therapeuticmodality,” Biochimica et Biophysica Acta 1844 (2014) 1943-1950; thecontents of each of which are hereby incorporated by reference. Itshould be appreciated, however, that the methods for making sweepingantibodies as provided herein are not meant to be limiting. Thus,additional methods for making sweeping antibodies are within the scopeof this disclosure.

Some aspects of the disclosure are based on the recognition that theaffinity (e.g., as expressed as Kd) of any of theanti-pro/latent-Myostatin antibodies provided herein are sensitive tochanges in pH. In some embodiments, the antibodies provided herein havean increased Kd of binding to pro/latent-Myostatin at a relatively lowpH (e.g., a pH ranging from 4.0-6.5) as compared to a relatively high pH(e.g., a pH ranging from 7.0-7.4). In some embodiments, the antibodiesprovided herein have a Kd of binding to pro/latent-Myostatin rangingfrom 10⁻³ M, 10⁻⁴ M, 10⁻⁵ M, 10⁻⁶ M, 10⁻⁷ M, 10⁻⁸ M when the pH isbetween 4.0 and 6.5. In some embodiments, the antibodies provided hereinhave a Kd of binding to pro/latent-Myostatin ranging from 10⁻⁶ M, 10⁻⁷M, 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M when the pH is between 7.0 and 7.4.In some embodiments, the antibodies provided herein have a Kd of bindingto pro/latent-Myostatin that is at least 2 fold, at least 10 fold, atleast 50 fold, at least 100 fold, at least 500 fold, at least 1000 fold,at least 5000 fold, or at least 10000 fold greater at a pH between 4.0and 6.5 as compared to a pH between 7.0 and 7.4.

In some embodiments, pro/latent-Myostatin antibodies are provided hereinthat do not specifically bind to an epitope within the amino acidsequence set forth as (SEQ ID NO: 64). In some embodiments,pro/latent-Myostatin antibodies provided herein do not specifically bindto the same epitope as an antibody described in Table 2a, 11a, 11b, or13 of International Patent Application Publication No. WO 2016/098357,which was published on Jun. 23, 2016, and which is based onInternational Patent Application No. PCT/JP2015/006323, which was filedon Dec. 18, 2015. In some embodiments, pro/latent-Myostatin antibodiesprovided herein do not compete or do not cross-compete for binding tothe same epitope as an antibody described in Table 2a, 11a, 11b, or 13of International Patent Application Publication No. WO 2016/098357,which was published on Jun. 23, 2016, and which is based onInternational Patent Application No. PCT/JP2015/006323, which was filedon Dec. 18, 2015. In some embodiments, pro/latent-Myostatin antibodiesprovided herein do not specifically bind to the same epitope as anantibody comprising a VH and a VL pair described in Table 2a, 11a, 11b,or 13 of International Patent Application Publication No. WO2016/098357, which was published on Jun. 23, 2016, and which is based onInternational Patent Application No. PCT/JP2015/006323, which was filedon Dec. 18, 2015. In some embodiments, pro/latent-Myostatin antibodiesprovided herein do not compete or do not cross-compete for binding tothe same epitope as an antibody comprising a VH and a VL pair describedin Table 2a, 11a, 11 b, or 13 of International Patent ApplicationPublication No. WO 2016/098357, which was published on Jun. 23, 2016,and which is based on International Patent Application No.PCT/JP2015/006323, which was filed on Dec. 18, 2015.

Polypeptides

Some aspects of the disclosure relate to a polypeptide having a sequenceselected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 25, SEQID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO 29. In someembodiments, the polypeptide is a variable heavy chain domain. In someembodiments, the polypeptide is at least 75% (e.g., 80%, 85%, 90%, 95%,98%, or 99%) identical to any one of the amino acid sequences set forthin SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ IDNO: 28, or SEQ ID NO 29.

Some aspects of the disclosure relate to a polypeptide having a sequenceselected from the group consisting of SEQ ID NO: 30, SEQ ID NO: 31, SEQID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO 35. In someembodiments, the polypeptide is a variable light chain domain. In someembodiments, the polypeptide is at least 75% (e.g., 80%, 85%, 90%, 95%,98%, or 99%) identical to any one of the amino acid sequences set forthin SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ IDNO: 34, or SEQ ID NO 35.

Antibodies that Compete with Anti-Pro/Latent-Myostatin Antibodies

Aspects of the disclosure relate to antibodies that compete orcross-compete with any of the antibodies provided herein. The term“compete”, as used herein with regard to an antibody, means that a firstantibody binds to an epitope of a protein (e.g., latent Myostatin) in amanner sufficiently similar to the binding of a second antibody, suchthat the result of binding of the first antibody with its epitope isdetectably decreased in the presence of the second antibody compared tothe binding of the first antibody in the absence of the second antibody.The alternative, where the binding of the second antibody to its epitopeis also detectably decreased in the presence of the first antibody, can,but need not be the case. That is, a first antibody can inhibit thebinding of a second antibody to its epitope without that second antibodyinhibiting the binding of the first antibody to its respective epitope.However, where each antibody detectably inhibits the binding of theother antibody with its epitope or ligand, whether to the same, greater,or lesser extent, the antibodies are said to “cross-compete” with eachother for binding of their respective epitope(s). Both competing andcross-competing antibodies are within the scope of this disclosure.Regardless of the mechanism by which such competition orcross-competition occurs (e.g., steric hindrance, conformational change,or binding to a common epitope, or portion thereof), the skilled artisanwould appreciate that such competing and/or cross-competing antibodiesare encompassed and can be useful for the methods and/or compositionsprovided herein.

Aspects of the disclosure relate to antibodies that compete orcross-compete with any of the antibodies provided herein. In someembodiments, an antibody binds at or near the same epitope as any of theantibodies provided herein. In some embodiments, an antibody binds nearan epitope if it binds within 15 or fewer amino acid residues of theepitope. In some embodiments, any of the antibodies provided herein bindwithin 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acidresidues of an epitope that is bound by any of the antibodies providedherein.

In another embodiment, an antibody competes or cross-competes forbinding to any of the antigens provided herein (e.g.,pro/latent-Myostatin) with an equilibrium dissociation constant, Kd,between the antibody and the protein of less than 10⁻⁶ M. In otherembodiments, an antibody competes or cross-competes for binding to anyof the antigens provided herein with a Kd in a range from 10⁻¹¹ M to10⁻⁶ M.

Aspects of the disclosure relate to antibodies that compete for bindingto pro/latent-Myostatin with any of the antibodies provided herein. Insome embodiments, the antibody binds to pro/latent-Myostatin at the sameepitope as any of the antibodies provided herein. For example, in someembodiments any of the antibodies provided herein bind at or near atolloid cleavage site or at or near a tolloid docking site ofpro/latent-Myostatin. In other embodiments, any of the antibodiesprovided herein bind at or near a proprotein convertase cleavage site orat or near a proprotein convertase docking site of pro/latent-Myostatin.In another embodiment, an antibody competes for binding topro/latent-Myostatin with an equilibrium dissociation constant, Kd,between the antibody and pro/latent-Myostatin of less than 10⁻⁶ M. Inother embodiments, the an antibody that competes with any of theantibodies provided herein binds to pro/latent-Myostatin with a Kd inranging from 10⁻¹¹ M to 10⁻⁶ M.

Any of the antibodies provided herein can be characterized using anysuitable methods. For example, one method is to identify the epitope towhich the antigen binds, or “epitope mapping.” There are many suitablemethods for mapping and characterizing the location of epitopes onproteins, including solving the crystal structure of an antibody-antigencomplex, competition assays, gene fragment expression assays, andsynthetic peptide-based assays, as described, for example, in Chapter 11of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1999. In anadditional example, epitope mapping can be used to determine thesequence to which an antibody binds. The epitope can be a linearepitope, i.e., contained in a single stretch of amino acids, or aconformational epitope formed by a three-dimensional interaction ofamino acids that may not necessarily be contained in a single stretch(primary structure linear sequence). Peptides of varying lengths (e.g.,at least 4-6 amino acids long) can be isolated or synthesized (e.g.,recombinantly) and used for binding assays with an antibody. In anotherexample, the epitope to which the antibody binds can be determined in asystematic screen by using overlapping peptides derived from the targetantigen sequence and determining binding by the antibody. According tothe gene fragment expression assays, the open reading frame encoding thetarget antigen is fragmented either randomly or by specific geneticconstructions and the reactivity of the expressed fragments of theantigen with the antibody to be tested is determined. The gene fragmentsmay, for example, be produced by PCR and then transcribed and translatedinto protein in vitro, in the presence of radioactive amino acids. Thebinding of the antibody to the radioactively labeled antigen fragmentsis then determined by immunoprecipitation and gel electrophoresis.Certain epitopes can also be identified by using large libraries ofrandom peptide sequences displayed on the surface of phage particles(phage libraries). Alternatively, a defined library of overlappingpeptide fragments can be tested for binding to the test antibody insimple binding assays. In an additional example, mutagenesis of anantigen binding domain, domain swapping experiments and alanine scanningmutagenesis can be performed to identify residues required, sufficient,and/or necessary for epitope binding. For example, domain swappingexperiments can be performed using a mutant of a target antigen in whichvarious fragments of the pro/latent-Myostatin polypeptide have beenreplaced (swapped) with sequences from a closely related, butantigenically distinct protein, such as another member of the TGFβprotein family (e.g., GDF11). By assessing binding of the antibody tothe mutant pro/latent-Myostatin, the importance of the particularantigen fragment to antibody binding can be assessed.

Alternatively, competition assays can be performed using otherantibodies known to bind to the same antigen to determine whether anantibody binds to the same epitope as the other antibodies. Competitionassays are well known to those of skill in the art. Any of the suitablemethods, e.g., the epitope mapping methods as described herein, can beapplied to determine whether an anti-pro/latent-Myostatin antibody bindsone or more of the specific residues/segments in pro/latent-Myostatin asdescribed herein. Further, the interaction of the antibody with one ormore of those defined residues in pro/latent-Myostatin can be determinedby routine technology. For example, a crystal structure can bedetermined, and the distances between the residues inpro/latent-Myostatin and one or more residues in the antibody can bedetermined accordingly. Based on such distance, whether a specificresidue in pro/latent-Myostatin interacts with one or more residues inthe antibody can be determined. Further, suitable methods, such ascompetition assays and target mutagenesis assays can be applied todetermine the preferential binding of a candidateanti-pro/latent-Myostatin antibody to pro/latent-Myostatin as comparedto another target such as a mutant pro/latent-Myostatin.

Production of Antibodies that Bind Pro/Latent-Myostatin

Numerous methods may be used for obtaining antibodies, or antigenbinding fragments thereof, of the disclosure. For example, antibodiescan be produced using recombinant DNA methods. Monoclonal antibodies mayalso be produced by generation of hybridomas (see e.g., Kohler andMilstein (1975) Nature, 256: 495-499) in accordance with known methods.Hybridomas formed in this manner are then screened using standardmethods, such as enzyme-linked immunosorbent assay (ELISA) and surfaceplasmon resonance (e.g., OCTET or BIACORE) analysis, to identify one ormore hybridomas that produce an antibody that specifically binds to aspecified antigen. Any form of the specified antigen may be used as theimmunogen, e.g., recombinant antigen, naturally occurring forms, anyvariants or fragments thereof, as well as antigenic peptide thereof(e.g., any of the epitopes described herein as a linear epitope orwithin a scaffold as a conformational epitope). One exemplary method ofmaking antibodies includes screening protein expression libraries thatexpress antibodies or fragments thereof (e.g., scFv), e.g., phage orribosome display libraries. Phage display is described, for example, inLadner et al., U.S. Pat. No. 5,223,409; Smith (1985) Science228:1315-1317; Clackson et al. (1991) Nature, 352: 624-628; Marks et al.(1991) J. Mol. Biol., 222: 581-597 WO92/18619; WO 91/17271; WO 92/20791;WO 92/15679; WO 93/01288; WO 92/01047; WO 92/09690; and WO 90/02809.

In addition to the use of display libraries, the specified antigen(e.g., proMyostatin) can be used to immunize a non-human animal, e.g., arodent, e.g., a mouse, hamster, or rat. In one embodiment, the non-humananimal is a mouse.

In another embodiment, a monoclonal antibody is obtained from thenon-human animal, and then modified, e.g., chimeric, using suitablerecombinant DNA techniques. A variety of approaches for making chimericantibodies have been described. See e.g., Morrison et al., Proc. Natl.Acad. Sci. U.S.A. 81:6851, 1985; Takeda et al., Nature 314:452, 1985,Cabilly et al., U.S. Pat. No. 4,816,567; Boss et al., U.S. Pat. No.4,816,397; Tanaguchi et al., European Patent Publication EP171496;European Patent Publication 0173494, United Kingdom Patent GB 2177096B.

For additional antibody production techniques, see Antibodies: ALaboratory Manual, eds. Harlow et al., Cold Spring Harbor Laboratory,1988. The present disclosure is not necessarily limited to anyparticular source, method of production, or other specialcharacteristics of an antibody.

Some aspects of the present disclosure relate to host cells transformedwith a polynucleotide or vector. Host cells may be a prokaryotic oreukaryotic cell. The polynucleotide or vector which is present in thehost cell may either be integrated into the genome of the host cell orit may be maintained extrachromosomally. The host cell can be anyprokaryotic or eukaryotic cell, such as a bacterial, insect, fungal,plant, animal or human cell. In some embodiments, fungal cells are, forexample, those of the genus Saccharomyces, in particular those of thespecies S. cerevisiae. The term “prokaryotic” includes all bacteriawhich can be transformed or transfected with a DNA or RNA molecules forthe expression of an antibody or the corresponding immunoglobulinchains. Prokaryotic hosts may include gram negative as well as grampositive bacteria such as, for example, E. coli, S. typhimurium,Serratia marcescens and Bacillus subtilis. The term “eukaryotic”includes yeast, higher plants, insects and vertebrate cells, e.g.,mammalian cells, such as NSO and CHO cells. Depending upon the hostemployed in a recombinant production procedure, the antibodies orimmunoglobulin chains encoded by the polynucleotide may be glycosylatedor may be non-glycosylated. Antibodies or the correspondingimmunoglobulin chains may also include an initial methionine amino acidresidue.

In some embodiments, once a vector has been incorporated into anappropriate host, the host may be maintained under conditions suitablefor high level expression of the nucleotide sequences, and, as desired,the collection and purification of the immunoglobulin light chains,heavy chains, light/heavy chain dimers or intact antibodies, antigenbinding fragments or other immunoglobulin forms may follow; see,Beychok, Cells of Immunoglobulin Synthesis, Academic Press, N.Y.,(1979). Thus, polynucleotides or vectors are introduced into the cellswhich in turn produce the antibody or antigen binding fragments.Furthermore, transgenic animals, preferably mammals, comprising theaforementioned host cells may be used for the large scale production ofthe antibody or antibody fragments.

The transformed host cells can be grown in fermenters and cultured usingany suitable techniques to achieve optimal cell growth. Once expressed,the whole antibodies, their dimers, individual light and heavy chains,other immunoglobulin forms, or antigen binding fragments, can bepurified according to standard procedures of the art, including ammoniumsulfate precipitation, affinity columns, column chromatography, gelelectrophoresis and the like; see, Scopes, “Protein Purification”,Springer Verlag, N.Y. (1982). The antibody or antigen binding fragmentscan then be isolated from the growth medium, cellular lysates, orcellular membrane fractions. The isolation and purification of the,e.g., microbially expressed antibodies or antigen binding fragments maybe by any conventional means such as, for example, preparativechromatographic separations and immunological separations such as thoseinvolving the use of monoclonal or polyclonal antibodies directed, e.g.,against the constant region of the antibody.

Aspects of the disclosure relate to a hybridoma, which provides anindefinitely prolonged source of monoclonal antibodies. As analternative to obtaining immunoglobulins directly from the culture ofhybridomas, immortalized hybridoma cells can be used as a source ofrearranged heavy chain and light chain loci for subsequent expressionand/or genetic manipulation. Rearranged antibody genes can be reversetranscribed from appropriate mRNAs to produce cDNA. In some embodiments,heavy chain constant region can be exchanged for that of a differentisotype or eliminated altogether. The variable regions can be linked toencode single chain Fv regions. Multiple Fv regions can be linked toconfer binding ability to more than one target or chimeric heavy andlight chain combinations can be employed. Any appropriate method may beused for cloning of antibody variable regions and generation ofrecombinant antibodies.

In some embodiments, an appropriate nucleic acid that encodes variableregions of a heavy and/or light chain is obtained and inserted into anexpression vectors which can be transfected into standard recombinanthost cells. A variety of such host cells may be used. In someembodiments, mammalian host cells may be advantageous for efficientprocessing and production. Typical mammalian cell lines useful for thispurpose include CHO cells, 293 cells, or NSO cells. The production ofthe antibody or antigen binding fragment may be undertaken by culturinga modified recombinant host under culture conditions appropriate for thegrowth of the host cells and the expression of the coding sequences. Theantibodies or antigen binding fragments may be recovered by isolatingthem from the culture. The expression systems may be designed to includesignal peptides so that the resulting antibodies are secreted into themedium; however, intracellular production is also possible.

The disclosure also includes a polynucleotide encoding at least avariable region of an immunoglobulin chain of the antibodies describedherein. In some embodiments, the variable region encoded by thepolynucleotide comprises at least one complementarity determining region(CDR) of the VH and/or VL of the variable region of the antibodyproduced by any one of the above described hybridomas.

Polynucleotides encoding antibody or antigen binding fragments may be,e.g., DNA, cDNA, RNA or synthetically produced DNA or RNA or arecombinantly produced chimeric nucleic acid molecule comprising any ofthose polynucleotides either alone or in combination. In someembodiments, a polynucleotide is part of a vector. Such vectors maycomprise further genes such as marker genes which allow for theselection of the vector in a suitable host cell and under suitableconditions.

In some embodiments, a polynucleotide is operatively linked toexpression control sequences allowing expression in prokaryotic oreukaryotic cells. Expression of the polynucleotide comprisestranscription of the polynucleotide into a translatable mRNA. Regulatoryelements ensuring expression in eukaryotic cells, preferably mammaliancells, are well known to those skilled in the art. They may includeregulatory sequences that facilitate initiation of transcription andoptionally poly-A signals that facilitate termination of transcriptionand stabilization of the transcript. Additional regulatory elements mayinclude transcriptional as well as translational enhancers, and/ornaturally associated or heterologous promoter regions. Possibleregulatory elements permitting expression in prokaryotic host cellsinclude, e.g., the PL, Lac, Trp or Tac promoter in E. coli, and examplesof regulatory elements permitting expression in eukaryotic host cellsare the AOX1 or GAL1 promoter in yeast or the CMV-promoter,SV40-promoter, RSV-promoter (Rous sarcoma virus), CMV-enhancer,SV40-enhancer or a globin intron in mammalian and other animal cells.

Beside elements which are responsible for the initiation oftranscription such regulatory elements may also include transcriptiontermination signals, such as the SV40-poly-A site or the tk-poly-A site,downstream of the polynucleotide. Furthermore, depending on theexpression system employed, leader sequences capable of directing thepolypeptide to a cellular compartment or secreting it into the mediummay be added to the coding sequence of the polynucleotide and have beendescribed previously. The leader sequence(s) is (are) assembled inappropriate phase with translation, initiation and terminationsequences, and preferably, a leader sequence capable of directingsecretion of translated protein, or a portion thereof, into, forexample, the extracellular medium. Optionally, a heterologouspolynucleotide sequence can be used that encode a fusion proteinincluding a C- or N-terminal identification peptide imparting desiredcharacteristics, e.g., stabilization or simplified purification ofexpressed recombinant product.

In some embodiments, polynucleotides encoding at least the variabledomain of the light and/or heavy chain may encode the variable domainsof both immunoglobulin chains or only one. Likewise, a polynucleotidesmay be under the control of the same promoter or may be separatelycontrolled for expression. Furthermore, some aspects relate to vectors,particularly plasmids, cosmids, viruses and bacteriophages usedconventionally in genetic engineering that comprise a polynucleotideencoding a variable domain of an immunoglobulin chain of an antibody orantigen binding fragment; optionally in combination with apolynucleotide that encodes the variable domain of the otherimmunoglobulin chain of the antibody.

In some embodiments, expression control sequences are provided aseukaryotic promoter systems in vectors capable of transforming ortransfecting eukaryotic host cells, but control sequences forprokaryotic hosts may also be used. Expression vectors derived fromviruses such as retroviruses, vaccinia virus, adeno-associated virus,herpes viruses, or bovine papilloma virus, may be used for delivery ofthe polynucleotides or vector into targeted cell population (e.g., toengineer a cell to express an antibody or antigen binding fragment). Avariety of appropriate methods can be used to construct recombinantviral vectors. In some embodiments, polynucleotides and vectors can bereconstituted into liposomes for delivery to target cells. The vectorscontaining the polynucleotides (e.g., the heavy and/or light variabledomain(s) of the immunoglobulin chains encoding sequences and expressioncontrol sequences) can be transferred into the host cell by suitablemethods, which vary depending on the type of cellular host.

Modifications

Antibodies or antigen binding fragments of the disclosure may bemodified with a detectable label, including, but not limited to, anenzyme, prosthetic group, fluorescent material, luminescent material,bioluminescent material, radioactive material, positron emitting metal,nonradioactive paramagnetic metal ion, and affinity label for detectionand isolation of pro/latent-Myostatin. The detectable substance may becoupled or conjugated either directly to the polypeptides of thedisclosure or indirectly, through an intermediate (such as, for example,a linker) using suitable techniques. Non-limiting examples of suitableenzymes include horseradish peroxidase, alkaline phosphatase,β-galactosidase, glucose oxidase, or acetylcholinesterase; non-limitingexamples of suitable prosthetic group complexes includestreptavidin/biotin and avidin/biotin; non-limiting examples of suitablefluorescent materials include biotin, umbelliferone, fluorescein,fluorescein isothiocyanate, rhodamine, dichlorotriazinylaminefluorescein, dansyl chloride, or phycoerythrin; an example of aluminescent material includes luminol; non-limiting examples ofbioluminescent materials include luciferase, luciferin, and aequorin;and examples of suitable radioactive material include a radioactivemetal ion, e.g., alpha-emitters or other radioisotopes such as, forexample, iodine (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I) carbon (¹⁴C), sulfur (³⁵S),tritium (³H), indium (¹¹⁵mIn, ¹¹³mIn, ¹¹²In, ¹¹¹In), and technetium(⁹⁹Tc, ⁹⁹mTc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium(¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, Lu,¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ⁸⁶R, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh,⁹⁷Ru, ⁶⁸Ge, ⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr, ³²P, ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn, ⁷⁵Se, andtin (¹¹³Sn, ¹¹⁷Sn). The detectable substance may be coupled orconjugated either directly to the anti-pro/latent-Myostatin antibodiesof the disclosure or indirectly, through an intermediate (such as, forexample, a linker) using suitable techniques. Anti-pro/latent-Myostatinantibodies conjugated to a detectable substance may be used fordiagnostic assays as described herein.

Pharmaceutical Compositions

One or more of the anti-pro/latent-Myostatin antibodies can be mixedwith a pharmaceutically acceptable carrier (excipient), includingbuffer, to form a pharmaceutical composition for use in alleviating adisease or disorder that is associated with myopathy. “Acceptable” meansthat the carrier must be compatible with the active ingredient of thecomposition (and preferably, capable of stabilizing the activeingredient) and not deleterious to the subject to be treated. Examplesof pharmaceutically acceptable excipients (carriers), including buffers,would be apparent to the skilled artisan and have been describedpreviously. See, e.g., Remington: The Science and Practice of Pharmacy20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover. Inone example, a pharmaceutical composition described herein contains morethan one anti-pro/latent-Myostatin antibodies that recognize differentepitopes/residues of the target antigen.

The pharmaceutical compositions to be used in the present methods cancomprise pharmaceutically acceptable carriers, excipients, orstabilizers in the form of lyophilized formulations or aqueoussolutions. (Remington: The Science and Practice of Pharmacy 20th Ed.(2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover). Acceptablecarriers, excipients, or stabilizers are nontoxic to recipients at thedosages and concentrations used, and may comprise buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrans; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).Pharmaceutically acceptable excipients are further described herein.

In some examples, the pharmaceutical composition described hereincomprises liposomes containing the anti-pro/latent-Myostatin antibody,which can be prepared by any suitable method, such as described inEpstein, et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang, etal., Proc. Natl. Acad. Sci. USA 77:4030 (1980); and U.S. Pat. Nos.4,485,045 and 4,544,545. Liposomes with enhanced circulation time aredisclosed in U.S. Pat. No. 5,013,556. Particularly useful liposomes canbe generated by the reverse phase evaporation method with a lipidcomposition comprising phosphatidylcholine, cholesterol andPEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes areextruded through filters of defined pore size to yield liposomes withthe desired diameter.

The anti-pro/latent-Myostatin antibody may also be entrapped inmicrocapsules prepared, for example, by coacervation techniques or byinterfacial polymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Exemplary techniques have beendescribed previously, see, e.g., Remington, The Science and Practice ofPharmacy 20th Ed. Mack Publishing (2000).

In other examples, the pharmaceutical composition described herein canbe formulated in sustained-release format. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(v nylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and 7ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), sucrose acetate isobutyrate, andpoly-D-(−)-3-hydroxybutyric acid.

The pharmaceutical compositions to be used for in vivo administrationmust be sterile. This is readily accomplished by, for example,filtration through sterile filtration membranes. Therapeutic antibodycompositions are generally placed into a container having a sterileaccess port, for example, an intravenous solution bag or vial having astopper pierceable by a hypodermic injection needle.

The pharmaceutical compositions described herein can be in unit dosageforms such as tablets, pills, capsules, powders, granules, solutions orsuspensions, or suppositories, for oral, parenteral or rectaladministration, or administration by inhalation or insufflation.

For preparing solid compositions such as tablets, the principal activeingredient can be mixed with a pharmaceutical carrier, e.g.,conventional tableting ingredients such as corn starch, lactose,sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalciumphosphate or gums, and other pharmaceutical diluents, e.g., water, toform a solid preformulation composition containing a homogeneous mixtureof a compound of the present disclosure, or a non-toxic pharmaceuticallyacceptable salt thereof. When referring to these preformulationcompositions as homogeneous, it is meant that the active ingredient isdispersed evenly throughout the composition so that the composition maybe readily subdivided into equally effective unit dosage forms such astablets, pills and capsules. This solid preformulation composition isthen subdivided into unit dosage forms of the type described abovecontaining from 0.1 mg to about 500 mg of the active ingredient of thepresent disclosure. The tablets or pills of the novel composition can becoated or otherwise compounded to provide a dosage form affording theadvantage of prolonged action. For example, the tablet or pill cancomprise an inner dosage and an outer dosage component, the latter beingin the form of an envelope over the former. The two components can beseparated by an enteric layer that serves to resist disintegration inthe stomach and permits the inner component to pass intact into theduodenum or to be delayed in release. A variety of materials can be usedfor such enteric layers or coatings, such materials including a numberof polymeric acids and mixtures of polymeric acids with such materialsas shellac, cetyl alcohol and cellulose acetate. Suitable surface-activeagents include, in particular, non-ionic agents, such aspolyoxyethylenesorbitans (e.g. Tween™ 20, 40, 60, 80 or 85) and othersorbitans (e.g. Span™ 20, 40, 60, 80 or 85). Compositions with asurface-active agent will conveniently comprise between 0.05 and 5%surface-active agent, and can be between 0.1 and 2.5%. It will beappreciated that other ingredients may be added, for example mannitol orother pharmaceutically acceptable vehicles, if necessary.

Suitable emulsions may be prepared using commercially available fatemulsions, such as Intralipid™, Liposyn™, Infonutrol™, Lipofundin™ andLipiphysan™. The active ingredient may be either dissolved in apre-mixed emulsion composition or alternatively it may be dissolved inan oil (e.g. soybean oil, safflower oil, cottonseed oil, sesame oil,corn oil or almond oil) and an emulsion formed upon mixing with aphospholipid (e.g. egg phospholipids, soybean phospholipids or soybeanlecithin) and water. It will be appreciated that other ingredients maybe added, for example glycerol or glucose, to adjust the tonicity of theemulsion. Suitable emulsions will typically contain up to 20% oil, forexample, between 5 and 20%.

The emulsion compositions can be those prepared by mixing ananti-proMyostatin antibody with Intralipid™ or the components thereof(soybean oil, egg phospholipids, glycerol and water).

Pharmaceutical compositions for inhalation or insufflation includesolutions and suspensions in pharmaceutically acceptable, aqueous ororganic solvents, or mixtures thereof, and powders. The liquid or solidcompositions may contain suitable pharmaceutically acceptable excipientsas set out above. In some embodiments, the compositions are administeredby the oral or nasal respiratory route for local or systemic effect.Compositions in preferably sterile pharmaceutically acceptable solventsmay be nebulised by use of gases. Nebulised solutions may be breatheddirectly from the nebulising device or the nebulising device may beattached to a face mask, tent or intermittent positive pressurebreathing machine. Solution, suspension or powder compositions may beadministered, preferably orally or nasally, from devices which deliverthe formulation in an appropriate manner.

Use of Anti-Pro/Latent-Myostatin Antibodies for TreatingDiseases/Disorders

The anti-pro/latent-Myostatin antibodies described herein are effectivein treating a disease or disorder associated with myopathy. As usedherein, the term “myopathy” refers to a muscular disease in which themuscle fibers do not function properly, typically resulting in muscularweakness. Myopathies include muscular diseases that are neuromuscular ormusculoskeletal in nature. In some embodiments, the myopathy is aninherited myopathy. Inherited myopathies include, without limitation,dystrophies, myotonias, congenital myopathies (e.g., nemaline myopathy,multi/minicore myopathy, and centronuclear myopathy), mitochondrialmyopathies, familial periodic myopathies, inflammatory myopathies andmetabolic myopathies (e.g., glycogen storage diseases and lipid storagedisorder). In some embodiments, the myopathy is an acquired myopathy.Acquired myopathies include, without limitation, external substanceinduced myopathy (e.g., drug-induced myopathy and glucocorticoidmyopathy, alcoholic myopathy, and myopathy due to other toxic agents),myositis (e.g., dermatomyositis, polymositis and inclusion bodymyositis), myositis ossificans, rhabdomyolysis, and myoglobinurias, anddisuse atrophy. In some embodiments, the myopathy is disuse atrophy,which may be caused by bone fracture (e.g. a hip fracture) or by nerveinjury (e.g., spinal cord injury (SCI)). In some embodiments themyopathy is related to a disease or disorder such as amyotrophic lateralsclerosis (ALS), spinal muscular atrophy (SMA), cachexia syndromes dueto renal failure, AIDS, cardiac conditions and/or cancer. In someembodiments the myopathy is related to ageing.

An aspect of the disclosure includes a method of treating a subjecthaving a myopathy, the method comprising administering to the subject aneffective amount of an antibody described above. In some embodiments,the myopathy is a primary myopathy. In another embodiment, the primarymyopathy comprises disuse atrophy. In other embodiments, the disuseatrophy is associated with hip fracture, elective joint replacement,critical care myopathy, spinal cord injury or stroke. In someembodiments, the myopathy is a secondary myopathy, in which muscle lossis secondary to a disease pathology. In other embodiments, the secondarymyopathy comprises denervation, genetic muscle weakness or cachexia. Inanother embodiment, the secondary myopathy is a denervation associatedwith amyotrophic lateral sclerosis or spinal muscular atrophy. In someembodiments, the secondary myopathy is a genetic muscle weaknessassociated with a muscular dystrophy. In other embodiments, thesecondary myopathy is a cachexia associated with renal failure, AIDS, acardiac condition, cancer or aging.

Another aspect of the disclosure includes a method of treating a subjecthaving a disease or condition related to aging. Exemplary diseases andconditions related to ageing include, without limitation, sarcopenia(age-related muscle loss), frailty, and androgen deficiency.

Another aspect of the disclosure includes a method of treating a subjecthaving a disease or condition related to disuse atrophy/trauma.Exemplary diseases and conditions related to disuse atrophy/traumainclude, without limitation, muscle weakness related to time spent in anintensive care unit (ICU), hip/joint replacement, hip fracture, stroke,bed rest, SCI, rotator cuff injury, knee replacement, bone fracture, andburns.

Another aspect of the disclosure includes a method of treating a subjecthaving a neurodegenerative disease or condition. Exemplaryneurodegenerative diseases or conditions include, without limitation,spinal muscular atrophy and amyotrophic lateral sclerosis (ALS).

Another aspect of the disclosure includes a method of treating a subjecthaving a disease or condition related to Cachexia. Exemplary diseasesand conditions related to cachexia include, without limitation, cancer,chronic heart failure, acquired immune deficiency syndrome (AIDS),chronic obstructive pulmonary disease (COPD), and chronic kidney disease(CKD).

Another aspect of the disclosure includes a method of treating a subjecthaving a disease or condition related to rare diseases. Exemplary rarediseases and conditions include, without limitation, osteogenesisimperfecta, sporadic Inclusion body myositis, and acute lymphoblasticleukemia.

Another aspect of the disclosure includes a method of treating a subjecthaving a disease or condition related to a metabolic disorder and/orbody composition. In some embodiments, the disease or condition isobesity (e.g., severe obesity), Prader-Willi, type II diabetes, oranorexia. However, additional diseases or conditions related tometabolic disorders and/or body composition would be apparent to theskilled artisan and are within the scope of this disclosure.

Another aspect of the disclosure includes a method of treating a subjecthaving a disease or condition related to congenital myopathies.Exemplary congenital myopathies include, without limitation, X-linkedmyotubular myopathy, autosomal dominant centronuclear myopathy,autosomal recessive centronuclear myopathy, nemaline myopathy, andcongenital fiber-type disproportion myopathy.

Another aspect of the disclosure includes a method of treating a subjecthaving a disease or condition related to muscular dystrophies. Exemplarymuscular dystrophies include, without limitation, Duchenne's, Becker's,facioscapulohumeral (FSH), and Limb-Girdle muscular dystrophies.

Another aspect of the disclosure includes a method of treating a subjecthaving a urogynecological related disease or condition, glotticdisorders (stenosis), extraocular myopathy, carpel tunnel,Guillain-Barré, or osteosarcoma.

To practice the method disclosed herein, an effective amount of thepharmaceutical composition described above can be administered to asubject (e.g., a human) in need of the treatment via a suitable route,such as intravenous administration, e.g., as a bolus or by continuousinfusion over a period of time, by intramuscular, intraperitoneal,intracerebrospinal, subcutaneous, intra-articular, intrasynovial,intrathecal, oral, inhalation or topical routes. Commercially availablenebulizers for liquid formulations, including jet nebulizers andultrasonic nebulizers are useful for administration. Liquid formulationscan be directly nebulized and lyophilized powder can be nebulized afterreconstitution. Alternatively, anti-pro/latent-Myostatin antibodies canbe aerosolized using a fluorocarbon formulation and a metered doseinhaler, or inhaled as a lyophilized and milled powder.

The subject to be treated by the methods described herein can be amammal, more preferably a human. Mammals include, but are not limitedto, farm animals, sport animals, pets, primates, horses, dogs, cats,mice and rats. A human subject who needs the treatment may be a humanpatient having, at risk for, or suspected of having a disease/disorderassociated with myopathy, such as those noted above. A subject having apro/latent-Myostatin-associated disease or disorder can be identified byroutine medical examination, e.g., laboratory tests, organ functionaltests, CT scans, or ultrasounds. A subject suspected of having any ofsuch disease/disorder might show one or more symptoms of thedisease/disorder. A subject at risk for the disease/disorder can be asubject having one or more of the risk factors for thatdisease/disorder.

“An effective amount” as used herein refers to the amount of each activeagent required to confer therapeutic effect on the subject, either aloneor in combination with one or more other active agents. Effectiveamounts vary, as recognized by those skilled in the art, depending onthe particular condition being treated, the severity of the condition,the individual patient parameters including age, physical condition,size, gender and weight, the duration of the treatment, the nature ofconcurrent therapy (if any), the specific route of administration andlike factors within the knowledge and expertise of the healthpractitioner. These factors are well known to those of ordinary skill inthe art and can be addressed with no more than routine experimentation.It is generally preferred that a maximum dose of the individualcomponents or combinations thereof be used, that is, the highest safedose according to sound medical judgment. It will be understood by thoseof ordinary skill in the art, however, that a patient may insist upon alower dose or tolerable dose for medical reasons, psychological reasonsor for virtually any other reasons. In some embodiments, an effectiveamount refers to the amount of an antibody, or antigen-binding portionthereof, which is sufficient to reduce or ameliorate the severity and/orduration of a disorder or one or more symptoms thereof, prevent theadvancement of a disorder, cause regression of a disorder, prevent therecurrence, development, onset or progression of one or more symptomsassociated with a disorder, detect a disorder, or enhance or improve theprophylactic or therapeutic effect(s) of another therapy (e.g.,prophylactic or therapeutic agent).

In some embodiments, in the context of administration of apro/latent-Myostatin antibody to a subject, an effective amount is anamount effective to increase mass of a target muscle in the subjectcompared with a control muscle mass. In some embodiments, the increasein muscle mass is an increase of at least 1.1-fold, at least 1.2-fold,at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, atleast 2-fold, at least 4-fold, at least 5-fold or more compared with acontrol muscle mass. In some embodiments, the increase in muscle mass isan increase in a range of 1-fold to 5-fold, 2-fold to 10-fold, 1-fold to1.5-fold, 1-fold to 2-fold, etc. compared with a control muscle mass.

As used herein, the term “control muscle mass” refers to a referencestandard useful for evaluating effects of a condition (e.g., treatmentwith a pro/latent-Myostatin antibody) on the mass of a target muscle ina subject. In some embodiments, a control muscle mass is a predeterminedvalue. In some embodiments, a control muscle mass is experimentallydetermined. In some embodiments, a control muscle mass is the mass of atarget muscle in a subject who has not been administered thepro/latent-Myostatin antibody. In some embodiments, a control musclemass is the mass (e.g., the average mass) of a target muscle in apopulation of subjects who have not been administered thepro/latent-Myostatin antibody. In some embodiments, a control musclemass is the mass of a target muscle in a subject prior to (e.g.,immediately prior to) being administered the pro/latent-Myostatinantibody. In some embodiments, a control muscle mass is the mass of atarget muscle in a subject who has been administered, in place of thepro/latent-Myostatin antibody, a normal antibody (e.g., of the sameisotype as the pro/latent-Myostatin antibody) that has been obtainedfrom an animal that has not been exposed to the antigen to which thepro/latent-Myostatin antibody is directed. In some embodiments, acontrol muscle mass is the mass of a target muscle in a subject who hasbeen administered, in place of the pro/latent-Myostatin antibody, avehicle, e.g., saline.

In some embodiments, in the context of administration of apro/latent-Myostatin antibody to a subject, an effective amount is anamount effective to increase force generation capacity (e.g., a maximalforce generation as determined in vitro with a muscle lever systemadapted with a horizontal perfusion bath) of a target muscle in thesubject compared with a control force generation capacity. In someembodiments, the increase in force generation capacity is an increase ofat least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, atleast 1.8-fold, at least 1.9-fold, at least 2-fold, at least 4-fold, atleast 5-fold or more compared with a control force generation capacity.In some embodiments, the increase in force generation capacity is anincrease in a range of 1-fold to 5-fold, 2-fold to 10-fold, 1-fold to1.5-fold, 1-fold to 2-fold, etc. compared with a control forcegeneration capacity.

As used herein, the term “control force generation capacity” refers to areference standard useful for evaluating effects of a condition (e.g.,treatment with a pro/latent-Myostatin antibody) on the force generationcapacity of a muscle in a subject. In some embodiments, a control forcegeneration capacity is a predetermined value. In some embodiments, acontrol force generation capacity is experimentally determined. In someembodiments, a control force generation capacity is the force generationcapacity of a target muscle in a subject who has not been administeredthe pro/latent-Myostatin antibody. In some embodiments, a control forcegeneration capacity is the force generation capacity (e.g., the averageforce generation capacity) of a target muscle in a population ofsubjects who have not been administered the pro/latent-Myostatinantibody. In some embodiments, a control force generation capacity isthe force generation capacity of a target muscle in a subject prior to(e.g., immediately prior to) being administered the pro/latent-Myostatinantibody. In some embodiments, a control force generation capacity isthe force generation capacity of a target muscle in a subject who hasbeen administered, in place of the pro/latent-Myostatin antibody, anormal antibody (e.g., of the same isotype as the pro/latent-Myostatinantibody) that has been obtained from an animal that has not beenexposed to the antigen to which the pro/latent-Myostatin antibody isdirected. In some embodiments, a control force generation capacity isthe force generation capacity of a target muscle in a subject who hasbeen administered, in place of the pro/latent-Myostatin antibody, avehicle, e.g., saline.

Empirical considerations, such as the half-life, generally willcontribute to the determination of the dosage. For example, antibodiesthat are compatible with the human immune system, such as humanizedantibodies or fully human antibodies, may be used to prolong half-lifeof the antibody and to prevent the antibody being attacked by the host'simmune system. Frequency of administration may be determined andadjusted over the course of therapy, and is generally, but notnecessarily, based on treatment and/or suppression and/or ameliorationand/or delay of a disease/disorder associated with myopathy.Alternatively, sustained continuous release formulations of ananti-pro/latent-Myostatin may be appropriate. Various formulations anddevices for achieving sustained release would be apparent to the skilledartisan and are within the scope of this disclosure.

In one example, dosages for an anti-pro/latent-Myostatin antibody asdescribed herein may be determined empirically in individuals who havebeen given one or more administration(s) of the antibody. Individualsare given incremental dosages of the antagonist. To assess efficacy ofthe antagonist, an indicator of the disease/disorder can be followed.

Generally, for administration of any of the antibodies described herein,an initial candidate dosage can be about 2 mg/kg. For the purpose of thepresent disclosure, a typical daily dosage might range from about any of0.1 μg/kg to 3 μg/kg to 30 μg/kg to 300 μg/kg to 3 mg/kg, to 30 mg/kg to100 mg/kg or more, depending on the factors mentioned above. Forrepeated administrations over several days or longer, depending on thecondition, the treatment is sustained until a desired suppression ofsymptoms occurs or until sufficient therapeutic levels are achieved toalleviate a disease or disorder associated with pro/latent-Myostatin, ora symptom thereof. An exemplary dosing regimen comprises administeringan initial dose of about 2 mg/kg, followed by a weekly maintenance doseof about 1 mg/kg of the antibody, or followed by a maintenance dose ofabout 1 mg/kg every other week. However, other dosage regimens may beuseful, depending on the pattern of pharmacokinetic decay that thepractitioner wishes to achieve. For example, dosing from one-four timesa week is contemplated. In some embodiments, dosing ranging from about 3μg/mg to about 2 mg/kg (such as about 3 μg/mg, about 10 μg/mg, about 30μg/mg, about 100 μg/mg, about 300 μg/mg, about 1 mg/kg, and about 2mg/kg) may be used. In some embodiments, dosing frequency is once everyweek, every 2 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every7 weeks, every 8 weeks, every 9 weeks, or every 10 weeks; or once everymonth, every 2 months, or every 3 months, every 4 months, every 5months, every 6 months, every 8 months, every 10 months, every year, orlonger. The progress of this therapy is easily monitored by conventionaltechniques and assays. The dosing regimen (including the antibody used)can vary over time.

In some embodiments, for an adult patient of normal weight, dosesranging from about 0.3 to 5.00 mg/kg may be administered. The particulardosage regimen, e.g., dose, timing and repetition, will depend on theparticular individual and that individual's medical history, as well asthe properties of the individual agents (such as the half-life of theagent, and other relevant considerations).

For the purpose of the present disclosure, the appropriate dosage of ananti-pro/latent-Myostatin antibody will depend on the specific antibody(or compositions thereof) employed, the type and severity of thedisease/disorder, whether the antibody is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antagonist, and the discretion of the attendingphysician. In some embodiments, a clinician will administer ananti-pro/latent-Myostatin antibody, until a dosage is reached thatachieves the desired result. Administration of ananti-pro/latent-Myostatin antibody can be continuous or intermittent,depending, for example, upon the recipient's physiological condition,whether the purpose of the administration is therapeutic orprophylactic, and other factors known to skilled practitioners. Theadministration of an anti-pro/latent-Myostatin antibody may beessentially continuous over a preselected period of time or may be in aseries of spaced dose, e.g., either before, during, or after developinga disease or disorder associated with pro/latent-Myostatin.

As used herein, the term “treating” refers to the application oradministration of a composition including one or more active agents to asubject, who has a disease/disorder associated with myopathy, a symptomof the disease/disorder, or a predisposition toward thedisease/disorder, with the purpose to cure, heal, alleviate, relieve,alter, remedy, ameliorate, improve, or affect the disorder, the symptomof the disease, or the predisposition toward the disease/disorder.

Alleviating a disease/disorder associated with pro/latent-Myostatinincludes delaying the development or progression of the disease, orreducing disease severity. Alleviating the disease does not necessarilyrequire curative results. As used therein, “delaying” the development ofa disease/disorder associated with pro/latent-Myostatin means to defer,hinder, slow, retard, stabilize, and/or postpone progression of thedisease. This delay can be of varying lengths of time, depending on thehistory of the disease and/or individuals being treated. A method that“delays” or alleviates the development of a disease, or delays the onsetof the disease, is a method that reduces probability of developing oneor more symptoms of the disease in a given time frame and/or reducesextent of the symptoms in a given time frame, when compared to not usingthe method. Such comparisons are typically based on clinical studies,using a number of subjects sufficient to give a statisticallysignificant result.

“Development” or “progression” of a disease means initial manifestationsand/or ensuing progression of the disease. Development of the diseasecan be detectable and assessed using standard clinical techniques.However, development also refers to progression that may beundetectable. For purpose of this disclosure, development or progressionrefers to the biological course of the symptoms. “Development” includesoccurrence, recurrence, and onset. As used herein “onset” or“occurrence” of a disease/disorder associated with myopathy includesinitial onset and/or recurrence.

In some embodiments, the anti-pro/latent-Myostatin antibody describedherein is administered to a subject in need of the treatment at anamount sufficient to inhibit the proteolytic activation ofpro/latent-Myostatin to active Myostatin by at least 20% (e.g., 30%,40%, 50%, 60%, 70%, 80%, 90% or greater) in vivo. In other embodiments,an antibody is administered in an amount effective in reducing thepro/latent-Myostatin or latent Myostatin level by at least 20% (e.g.,30%, 40%, 50%, 60%, 70%, 80%, 90% or greater).

Conventional methods, known to those of ordinary skill in the art ofmedicine, can be used to administer the pharmaceutical composition tothe subject, depending upon the type of disease to be treated or thesite of the disease. This composition can also be administered via otherconventional routes, e.g., administered orally, parenterally, byinhalation spray, topically, rectally, nasally, buccally, vaginally orvia an implanted reservoir. The term “parenteral” as used hereinincludes subcutaneous, intracutaneous, intravenous, intramuscular,intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal,intralesional, and intracranial injection or infusion techniques. Inaddition, it can be administered to the subject via injectable depotroutes of administration such as using 1-, 3-, or 6-month depotinjectable or biodegradable materials and methods.

Injectable compositions may contain various carriers such as vegetableoils, dimethylactamide, dimethylformamide, ethyl lactate, ethylcarbonate, isopropyl myristate, ethanol, and polyols (glycerol,propylene glycol, liquid polyethylene glycol, and the like). Forintravenous injection, water soluble antibodies can be administered bythe drip method, whereby a pharmaceutical formulation containing theantibody and a physiologically acceptable excipients is infused.Physiologically acceptable excipients may include, for example, 5%dextrose, 0.9% saline, Ringer's solution or other suitable excipients.Intramuscular preparations, e.g., a sterile formulation of a suitablesoluble salt form of the antibody, can be dissolved and administered ina pharmaceutical excipient such as Water-for-Injection, 0.9% saline, or5% glucose solution.

In one embodiment, an anti-pro/latent-Myostatin antibody is administeredvia site-specific or targeted local delivery techniques. Examples ofsite-specific or targeted local delivery techniques include variousimplantable depot sources of the anti-pro/latent-Myostatin antibody orlocal delivery catheters, such as infusion catheters, an indwellingcatheter, or a needle catheter, synthetic grafts, adventitial wraps,shunts and stents or other implantable devices, site specific carriers,direct injection, or direct application. See, e.g., PCT Publication No.WO 00/53211 and U.S. Pat. No. 5,981,568.

Targeted delivery of therapeutic compositions containing apolynucleotide, or expression vector can also be used. Receptor-mediatedDNA delivery techniques are described in, for example, Findeis et al.,Trends Biotechnol. (1993) 11:202; Chiou et al., Gene Therapeutics:Methods And Applications Of Direct Gene Transfer (J. A. Wolff, ed.)(1994); Wu et al., J. Biol. Chem. (1988) 263:621; Wu et al., J. Biol.Chem. (1994) 269:542; Zenke et al., Proc. Natl. Acad. Sci. USA (1990)87:3655; Wu et al., J. Biol. Chem. (1991) 266:338.

Therapeutic compositions containing a polynucleotide (e.g., thoseencoding the anti-pro/latent-Myostatin antibodies described herein) areadministered in a range of about 100 ng to about 200 mg of DNA for localadministration in a gene therapy protocol. In some embodiments,concentration ranges of about 500 ng to about 50 mg, about 1 μg to about2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg of DNAor more can also be used during a gene therapy protocol.

The therapeutic polynucleotides and polypeptides described herein can bedelivered using gene delivery vehicles. The gene delivery vehicle can beof viral or non-viral origin (see generally, Jolly, Cancer Gene Therapy(1994) 1:51; Kimura, Human Gene Therapy (1994) 5:845; Connelly, HumanGene Therapy (1995) 1:185; and Kaplitt, Nature Genetics (1994) 6:148).Expression of such coding sequences can be induced using endogenousmammalian or heterologous promoters and/or enhancers. Expression of thecoding sequence can be either constitutive or regulated.

Suitable viral-based vectors for delivery of a desired polynucleotide(e.g., encoding an antibody disclosed herein) and expression in adesired cell are within the scope of this disclosure. Exemplaryviral-based vehicles include, but are not limited to, recombinantretroviruses (see, e.g., PCT Publication Nos. WO 90/07936; WO 94/03622;WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; WO 91/02805; U.S.Pat. Nos. 5,219,740 and 4,777,127; GB Patent No. 2,200,651; and EPPatent No. 0 345 242), alphavirus-based vectors (e.g., Sindbis virusvectors, Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross Rivervirus (ATCC VR-373; ATCC VR-1246) and Venezuelan equine encephalitisvirus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)), andadeno-associated virus (AAV) vectors (see, e.g., PCT Publication Nos. WO94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO95/00655). Administration of DNA linked to killed adenovirus asdescribed in Curiel, Hum. Gene Ther. (1992) 3:147 can also be employed.

Non-viral delivery vehicles and methods can also be employed, including,but not limited to, polycationic condensed DNA linked or unlinked tokilled adenovirus alone (see, e.g., Curiel, Hum. Gene Ther. (1992)3:147); ligand-linked DNA (see, e.g., Wu, J. Biol. Chem. (1989)264:16985); eukaryotic cell delivery vehicles cells (see, e.g., U.S.Pat. No. 5,814,482; PCT Publication Nos. WO 95/07994; WO 96/17072; WO95/30763; and WO 97/42338) and nucleic charge neutralization or fusionwith cell membranes. Naked DNA can also be employed. Exemplary naked DNAintroduction methods are described in PCT Publication No. WO 90/11092and U.S. Pat. No. 5,580,859. Liposomes that can act as gene deliveryvehicles are described in U.S. Pat. No. 5,422,120; PCT Publication Nos.WO 95/13796; WO 94/23697; WO 91/14445; and EP Patent No. 0524968.Additional approaches are described in Philip, Mol. Cell. Biol. (1994)14:2411, and in Woffendin, Proc. Natl. Acad. Sci. (1994) 91:1581.

The particular dosage regimen, e.g., dose, timing and repetition, usedin the method described herein will depend on the particular subject andthat subject's medical history.

In some embodiments, more than one anti-pro/latent-Myostatin antibodies,or a combination of an anti-pro/latent-Myostatin antibody and anothersuitable therapeutic agent, may be administered to a subject in need ofthe treatment. The antagonist can be the same type or different fromeach other. The anti-pro/latent-Myostatin antibody can also be used inconjunction with other agents that serve to enhance and/or complementthe effectiveness of the agents.

Treatment efficacy for a disease/disorder associated with myopathy canbe assessed using any suitable methods. For example, treatment efficacyfor a disease/disorder associated with myopathy can be assessed byevaluating muscle weakness (e.g., assessing the pattern and severity ofweakness), electromyography, evaluating blood chemistries (e.g.,assessing electrolytes, assessing endocrine causes, measuring creatininekinase level, determining erythrocyte sedimentation rate and performingantinuclear antibody assays), and evaluating biopsies (e.g., byhistologic, histochemical, electron microscopic, biochemical, andgenetic analysis).

Kits for Use in Alleviating Diseases/Disorders Associated with Myopathy

The present disclosure also provides kits for use in alleviatingdiseases/disorders associated with myopathy. Such kits can include oneor more containers comprising an anti-pro/latent-Myostatin antibody,e.g., any of those described herein.

In some embodiments, the kit can comprise instructions for use inaccordance with any of the methods described herein. The includedinstructions can comprise a description of administration of theanti-pro/latent-Myostatin antibody to treat, delay the onset, oralleviate a target disease as those described herein. The kit mayfurther comprise a description of selecting an individual suitable fortreatment based on identifying whether that individual has the targetdisease. In still other embodiments, the instructions comprise adescription of administering an antibody to an individual at risk of thetarget disease.

The instructions relating to the use of an anti-pro/latent-Myostatinantibody generally include information as to dosage, dosing schedule,and route of administration for the intended treatment. The containersmay be unit doses, bulk packages (e.g., multi-dose packages) or sub-unitdoses. Instructions supplied in the kits of the disclosure are typicallywritten instructions on a label or package insert (e.g., a paper sheetincluded in the kit), but machine-readable instructions (e.g.,instructions carried on a magnetic or optical storage disk) are alsoacceptable.

The label or package insert indicates that the composition is used fortreating, delaying the onset and/or alleviating a disease or disorderassociated with myopathy. Instructions may be provided for practicingany of the methods described herein.

The kits of this disclosure are in suitable packaging. Suitablepackaging includes, but is not limited to, vials, bottles, jars,flexible packaging (e.g., sealed Mylar or plastic bags), and the like.Also contemplated are packages for use in combination with a specificdevice, such as an inhaler, nasal administration device (e.g., anatomizer) or an infusion device such as a minipump. A kit may have asterile access port (for example the container may be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). The container may also have a sterile access port(for example the container may be an intravenous solution bag or a vialhaving a stopper pierceable by a hypodermic injection needle). At leastone active agent in the composition is an anti-pro/latent-Myostatinantibody as those described herein.

Kits may optionally provide additional components such as buffers andinterpretive information. Normally, the kit comprises a container and alabel or package insert(s) on or associated with the container. In someembodiments, the disclosure provides articles of manufacture comprisingcontents of the kits described above.

Assays for Detecting Pro/Latent-Myostatin

In some embodiments, methods and compositions provided herein relate toa method for detecting pro/latent-Myostatin in a sample obtained from asubject. As used herein, a “subject” refers to an individual organism,for example, an individual mammal. In some embodiments, the subject is ahuman. In some embodiments, the subject is a non-human mammal. In someembodiments, the subject is a non-human primate. In some embodiments,the subject is a rodent. In some embodiments, the subject is a sheep, agoat, a cattle, a cat, or a dog. In some embodiments, the subject is avertebrate, an amphibian, a reptile, a fish, an insect, a fly, or anematode. In some embodiments, the subject is a research animal. In someembodiments, the subject is genetically engineered, e.g., a geneticallyengineered non-human subject. The subject may be of either sex and atany stage of development. In some embodiments, the subject is a patientor a healthy volunteer.

In some embodiments, a method for detecting a pro/latent-Myostatin in asample obtained from a subject involves (a) contacting the sample withthe anti-pro/latent-Myostatin antibody under conditions suitable forbinding of the antibody to the antigen, if the antigen is present in thesample, thereby forming binding complexes; and (b) determining the levelof the antibody or antigen binding fragment bound to the antigen (e.g.,determining the level of the binding complexes).

As used herein a binding complex refers to a biomolecular complex ofantibody (including antigen binding fragments) bound to antigen (e.g.,pro/latent-Myostatin protein). Binding complexes may comprise antibodieswith a single specificity or two or more antibodies or antigen bindingfragments with different specificities. In one embodiment, a bindingcomplex comprises two or more antibodies recognizing different antigenicsites on the same antigen. In some instances, an antibody may be boundto an antigen, having bound to it other biomolecules such as RNA, DNA,polysaccharides or proteins. In one embodiment, a binding complexcomprises two or more antibodies recognizing different antigens. In someembodiments, an antibody in a binding complex (e.g., an immobilizedantibody bound to antigen), may itself by bound, as an antigen, to anantibody (e.g., a detectably labeled antibody). Thus, binding complexesmay, in some instances, comprise multiple antigens and multipleantibodies or antigen binding fragments.

Antigens present in binding complexes may or may not be in their nativein situ conformation. In some embodiments, a binding complex is formedbetween an antibody and a purified protein antigen, or isolated proteinscomprising antigen, in which the antigen is not in its native in situconformation. In some embodiments, a binding complex is formed betweenan antibody and a purified protein antigen, in which the antigen is notin its native in situ conformation and is immobilized on solid support(e.g., a PVDF membrane). In some embodiments, a binding complex isformed with an antibody and, for example, a cell surface protein that ispresent in situ in a native confirmation (e.g., on the surface of acell).

Antibodies in binding complexes may or may not be detectably labeled. Insome embodiments, binding complexes comprise detectably labeledantibodies and non-labeled antibodies. In some embodiments, bindingcomplexes comprise detectably labeled antigen. In some embodiments,antibodies, in binding complexes, are immobilized to one or more solidsupports. In some embodiments, antigens, in binding complexes, areimmobilized to one or more solid supports. Exemplary solid supports aredisclosed herein and will be apparent to one of ordinary skill in theart. The foregoing examples of binding complexes are not intended to belimiting. Other examples of binding complexes will be apparent to one orordinary skill in the art.

In any of the detection, diagnosis, and monitoring methods, theantibody, (including antigen binding fragments) or antigen may beconjugated to a solid support surface, either directly or indirectly.Methods for conjugation to solid supports are standard and can beaccomplished via covalent and non-covalent interactions. Non-limitingexamples of conjugation methods include: adsorption, cross-linking,protein A/G-antibody interactions, and streptavidin-biotin interactions.Other methods of conjugation will be readily apparent to one of ordinaryskill in the art.

In some aspects, detection, diagnosis, and monitoring methods includecomparing the level of the antibody (including antigen bindingfragments) bound to the antigen (e.g., pro/latent-Myostatin) to one ormore reference standards. The reference standard may be, for example,the level of a corresponding pro/latent-Myostatin in a subject that doesor does not have a pro/latent-Myostatin. In one embodiment, thereference standard is the level of pro/latent-Myostatin detected in asample that does not contain pro/latent-Myostatin (e.g., a backgroundlevel). Alternatively, a background level can be determined from asample that contains a particular pro/latent-Myostatin, by contactingthe sample with non-specific antibodies (e.g., antibodies obtained fromnon-immune serum). Then again, the reference standard may be the levelof pro/latent-Myostatin detected in a sample that does containpro/latent-Myostatin (e.g., a positive control). In some cases, thereference standard may be a series of levels associated with varyingconcentrations of pro/latent-Myostatin in a sample and useful forquantifying the concentration of pro/latent-Myostatin in the testsample. The foregoing examples of reference standards are not limitingand other suitable reference standard will be readily apparent to one ofordinary skill in the art. In some embodiments, the level of theantibody bound to pro/latent-Myostatin is compared to the level ofmature Myostatin. In some instances the level of pro/latent-Myostatin iscompared to mature Myostatin to determine the ratio of inactive toactive Myostatin in the sample.

The level of pro/latent-Myostatin may be measured, as provided herein,from a biological sample. A biological sample refers to any biologicalmaterial which may be obtained from a subject or cell. For example, abiological sample may be whole blood, plasma, serum, saliva,cerebrospinal fluid, urine, cells (or cell lysate) or tissue (e.g.,normal tissue or tumor tissue). In some embodiments, a biological sampleis a fluid sample. In some embodiments, a biological sample is a solidtissue sample. For example, a tissue sample may include, withoutlimitation skeletal muscle, cardiac muscle, adipose tissue as well astissue from other organs. In some embodiments, a biological sample is abiopsy sample. In some embodiments, a solid tissue sample may be madeinto a fluid sample using routine methods in the art.

A biological sample may also include one or more cells of a cell line.In some embodiments, a cell line includes human cells, primate cells(e.g., vero cells), rat cells (e.g., GH3 cells, OC23 cells) or mousecells (e.g., MC3T3 cells). There are a variety of human cell lines,including, without limitation, human embryonic kidney (HEK) cells, HeLacells, cancer cells from the National Cancer Institute's 60 cancer celllines (NCI60), DU145 (prostate cancer) cells, Lncap (prostate cancer)cells, MCF-7 (breast cancer) cells, MDA-MB-438 (breast cancer) cells,PC3 (prostate cancer) cells, T47D (breast cancer) cells, THP-1 (acutemyeloid leukemia) cells, U87 (glioblastoma) cells, SHSY5Y humanneuroblastoma cells (cloned from a myeloma) and Saos-2 (bone cancer)cells.

A further embodiment relates to a method for monitoring a disease, acondition, or any treatment thereof (e.g., myopathy or myopathytreatment) in a subject having, or at risk of having, the disease orcondition comprising: (a) obtaining a biological sample from thesubject, (b) determining the level of a pro/latent-Myostatin in thebiological sample using an antibody that detects pro/latent-Myostatin,and (c) repeating steps (a) and (b) on one or more occasions. Myostatinhas been used as a biomarker for muscle atrophy, however, the currentlyavailable commercial methods and reagents (e.g., antibodies used inELISAs and Western Blots) are either not specific for Myostatin, detectonly mature myostatin or do not detect myostatin at all. Thus, providedherein are methods and reagents (e.g., antibodies) for detectingpro/latent-Myostatin in the context of diseases and/or conditions (e.g.,muscle atrophy) for diagnostic purposes. As one example, the level ofpro/latent-Myostatin may be measured in a subject, or biological sampletherefrom, to detect or monitor the progression of a disease orcondition. As another example, the level of pro/latent-Myostatin may bemeasured in a subject, or biological sample therefrom, to monitor theresponse to a treatment for a disease or condition. It should beappreciated that the level of pro/latent-Myostatin may be monitored overany suitable period of time, which may differ depending on the diseaseor condition, the subject has or any treatment regimen that the subjectmay be subject to.

Another embodiment relates to a diagnostic composition comprising anyone of the above described antibodies, antigen binding fragments,polynucleotides, vectors or cells and optionally suitable means fordetection. The antibodies are, for example, suited for use inimmunoassays in which they can be utilized in liquid phase or bound to asolid phase carrier. Examples of immunoassays which can utilize theantibody are competitive and non-competitive immunoassays in either adirect or indirect format. Examples of such immunoassays are the EnzymeLinked Immunoassay (ELISA), radioimmunoassay (RIA), the sandwich(immunometric assay), flow cytometry, the western blot assay,immunoprecipitation assays, immunohistochemistry, immuno-microscopy,lateral flow immuno-chromatographic assays, and proteomics arrays. Theantigens and antibodies can be bound to many different solid supports(e.g., carriers, membrane, columns, proteomics array, etc.). Examples ofsolid support materials include glass, polystyrene, polyvinyl chloride,polyvinylidene difluoride, polypropylene, polyethylene, polycarbonate,dextran, nylon, amyloses, natural and modified celluloses, such asnitrocellulose, polyacrylamides, agaroses, and magnetite. The nature ofthe support can be either fixed or suspended in a solution (e.g.,beads).

By a further embodiment, antibodies (including antigen bindingfragments) provided herein may also be used in a method for evaluatingpro/latent-Myostatin expression in a subject by obtaining a biologicalsample from the subject which may be a tissue sample, a blood sample orany other appropriate body fluid sample. The procedure may comprisecontacting the blood sample (whole blood, serum, plasma), a tissuesample, or protein sample isolated therefrom, with an antibody, underconditions enabling the formation of binding complexes between antibodyand antigen. The level of such binding complexes may then be determinedby any suitable method. In some embodiments, the biological sample iscontacted with the antibody under conditions suitable for binding of theantibody to a pro/latent-Myostatin protein, if the antigen is present inthe sample, and formation of binding complexes consisting of antibody,bound to the antigen. This contacting step is typically performed in areaction chamber, such as a tube, plate well, membrane bath, cellculture dish, microscope slide, and the like. In some embodiments, anantibody is immobilized on a solid support. In some embodiments, theantigen is immobilized on a solid support. In some embodiments, thesolid support is the surface of the reaction chamber. In someembodiments, the solid support is of a polymeric membrane (e.g.,nitrocellulose strip, Polyvinylidene Difluoride (PVDF) membrane, etc.).Other appropriate solid supports may be used.

In some embodiments, an antibody is immobilized on the solid supportprior to contacting with the antigen. In other embodiments,immobilization of the antibody is performed after formation of bindingcomplexes. In still other embodiments, antigen is immobilized on a solidsupport prior to formation of binding complexes. A detection reagent isadded to the reaction chamber to detect immobilized binding complexes.In some embodiments, the detection reagent comprises a detectablylabeled secondary antibody directed against the antigen. In someembodiments, the primary antibody is itself detectable labeled, and isthereby the detection reagent.

In one aspect, detection methods comprise the steps of immobilizingantibodies to a solid support; applying a sample (e.g., a biologicalsample or isolated protein sample) to the solid support under conditionsthat permit binding of antigen to the antibodies, if present in thesample; removing the excess sample from the solid support; applyingdetectably labeled antibodies under conditions that permit binding ofthe detectably labeled antibodies to the antigen-bound immobilizedantibodies; washing the solid support and assaying for the presence oflabel on the solid support.

In some embodiments, the antigen is immobilized on the solid support,such as a PVDF membrane, prior to contacting with the antibody in areaction chamber (e.g., a membrane bath). A detection reagent is addedto the reaction chamber to detect immobilized binding complexes. In someembodiments, the detection reagent comprises a detectably labeledsecondary antibody directed against the antigen. In some embodiments,the detection reagent comprises a detectably labeled secondary antibodydirected against the primary antibody. As disclosed herein, thedetectable label may be, for example, a radioisotope, a fluorophore, aluminescent molecule, an enzyme, a biotin-moiety, an epitope tag, or adye molecule. In some embodiments, the primary antibody is itselfdetectable labeled, and is thereby the detection reagent. Suitabledetectable labels are described herein, and will be readily apparent toone of ordinary skill in the art.

Accordingly, diagnostic kits, suitable for home or clinical use (pointof care service), are provided that comprise (a) detectably labeledand/or non-labeled antibodies, as antigen binding reagents (e.g.,pro/latent-Myostatin binding reagents); (b) a detection reagent; and,optionally, (c) complete instructions for using the reagents to detectantigens in a sample. In some embodiments, the diagnostic kit includesthe antibody, and/or pro/latent-Myostatin immobilized on a solidsupport. Any of the solid supports described herein are suitable forincorporation in the diagnostic kits. In a preferred embodiment, thesolid support is the surface of a reaction chamber of a plate well.Typically, the plate well is in a multi-well plate having a number ofwells selected from: 6, 12, 24, 96, 384, and 1536, but it is not solimited. In other embodiments, the diagnostic kits provide a detectablylabeled antibody. Diagnostic kits are not limited to these embodimentsand other variations in kit composition will be readily apparent to oneof ordinary skill in the art.

The following specific embodiments are, therefore, to be construed asmerely illustrative, and not limitative of the remainder of thedisclosure in any way whatsoever. All publications cited herein areincorporated by reference for the purposes or subject matter referencedherein.

EXAMPLES Example 1: Generation and Selection of Antibodies AntibodySummary

Ab2 is a fully human anti-pro/latent-Myostatin monoclonal antibody ofthe IgG4/lambda isotype that binds to human pro- and latent-myostatinwith high affinity (Kd=3420 pM by ForteBio BLI). The antibody is capableof inhibiting the proteolytic activation of pro/latent-Myostatin withIC50 values in the 0.5 micromolar range (which is at or near the limitof the assay). The theoretical molecular weight of the polypeptide is144,736 Da and its theoretical pI is 6.7. Affinity optimization usingantibody display was performed to identify higher affinity variants Ab4and Ab6. The affinity optimized variants are similarly constructed onthe human IgG4/lambda isotype frameworks.

TABLE 2 Biochemical properties of candidate anti-Pro/latent-Myostatinantibodies Affinity Theoretical (Octet) MW (Da) Calculated Antibody pM*aglycosylated pI Ab1 4760 144809.8 6.9 Ab2 3420 144735.6 6.7 Ab4 472144661.7 6.7 Ab6 331 144629.5 6.7

Platform and Identification of Parental Antibody

The parental Ab1 antibody was identified via selection of a naïve phagedisplay library using pro- and latent-myostatin as the primary antigensfor selection. Phage selection and initial screening were performedusing a library displaying conventional scFv in a format similar to thatdescribed by McCafferty et al. (McCafferty et al., 1990). Each round ofselection consisted of pre-clearing (for removal of nonspecific phageantibodies), incubation with antigen, washing, elution andamplification. Selections were performed via multiple rounds using bothsolid phase (biotinylated antigens coated on immunotubes) and solutionphase (biotinylated antigens, captured using streptavidin coated beads)panning strategies.

In total, 10,000 individual scFv clones were screened for binding topro- or latent-myostatin through two separate campaigns. The firstprogram utilized pro/latent-Myostatin as an antigen, while a secondcampaign used latent Myostatin as an antigen. DNA for scFv clones ofinterest were sequenced and 216 unique clones were identified. Positivebinding scFv clones were counter-screened for binding to proGDF11 aswell as to a panel of unrelated proteins to confirm specificity forpro/latent-Myostatin. From the panel of unique scFv clones, 101 (of 134GDF8 specific clones) were converted to full length IgG (IgG1 isotype)for additional characterization.

Full-length IgG antibodies were further characterized by ELISA forbinding to the human and murine pro- and latent-forms of myostatin andGDF11. Antibodies were also screened for binding to the Myostatinprodomain, proTGFβ (human and murine), the mature growth factor ofMyostatin, the GDF11 mature growth factor, the Activin A growth factor,and proActivin A. Lead antibodies were selected based on theircross-reactivity with pro- and latent human and murine Myostatin, withno interactions with GDF11, Activin, or TGFβ proteins.

Two forms of epitope binning were employed. First, chimeric constructswhich swapped portions of the prodomains of Myostatin and GDF11 weredesigned and produced. These chimeric proteins were assayed forinteraction with screening antibodies by ELISA. Epitope binning wascarried out using a ForteBio BLI instrument, in which the biotinylatedpro/latent-Myostatin antibody was immobilized on a streptavidin coatedbiosensor chip, and cross-blocking of antibodies was evaluated by sensorresponse. These epitope binning experiments, along with data from theELISA binding experiments, allowed for the segregation of ourfunctionally active lead antibodies (see below) into three distinctepitope groups (see Table 3).

TABLE 3 Ranking of five anti-pro/latent-Myostatin IgG1 antibodies HumanMurine proGDF8 proGDF8 % body weight % lean mass proGDF8 IC50² (μM)IC50² (μM) increase increase Clone Kd (μM) Reporter Reporter in 6 weeksin 4 weeks Epitope ID (octet) assay assay 25 mg/kg/week 20 mg/kg/weekbin Ab1 11.5 0.996 1.46 14.58* 14.1* 1 Ab7 28 0.983 1.68 12.42* ND 1 Ab80.5 6.037 139¹    10.33*  7.4 2 Ab9 22 12.16 19.86  7.44 ND 3 Ab10 0.30.772 ND ND 14.3* 1 *Statistical significance by one-way ANOVA withDunnett. Ab8 does not bind latent myostatin, only proMyostatin. Murinepro/latent-Myostatin preparations have ~40% latent material whichreduces the apparent efficacy in functional assays. ND: Not determined.

In order to evaluate the ability of antibodies to bind and inhibit theactivation of pro/latent-Myostatin, a number of biochemical and cellularassays were established. Binding kinetics to pro- and latent-Myostatinwas measured by ForteBio Octet, in which the biotinylated substrateprotein was immobilized on streptavidin coated sensor chips. Theequilibrium dissociation constants of candidates from screening areshown in Table 3.

To measure the ability of the IgGs to inhibit Myostatin signaling, aMyostatin activation assay was developed. Conditioned medium from cellsoverexpressing either mTll2 (the tolloid protease require for Myostatinactivation) or Furin (the proprotein convertase which cleaves the maturegrowth factor from the prodomain) were produced. Followingpre-incubation with the test antibody, pro/latent-Myostatin or latentMyostatin was incubated with either a mixture of mTll2 and Furinconditioned media (proMyostatin) or mTll2 conditioned media (latentMyostatin). Following an overnight proteolysis reaction, the release ofmature growth factor was measured using a CAGA-based reporter assay in293T cells. Antibodies were further validated by dose response, in thesame assay, the results of which are shown in Table 3.

Five parental antibodies (Table 3) demonstrated consistently potentselectivity and activity in all of the above assays and were furtherchosen for further characterization in vivo (discussed in Example 2).For consistency, the binding and activity of these antibodies towardspro/latent-Myostatin is summarized, as Ab8 does not recognize latentmyostatin.

To determine the mechanism of action of antibody candidates, sampleswere analyzed by western blotting using a polyclonal antibody raisedagainst the prodomain of myostatin, as shown in FIG. 3. This allowed fortracking of a fragment (boxed) of the myostatin prodomain which isgenerated after mTll2 cleavage. A dose-dependent decrease was seen inthe generation of this fragment as the concentration of Ab1 isincreased. This experiment indicates that the antibodies in epitope bin1 act by blocking the cleavage of pro- and latent-myostatin by thetolloid family of proteases.

Based on the in vitro and in vivo activity of the activeanti-pro/latent-Myostatin antibodies, Ab1 was selected as the lead forfurther optimization, including affinity maturation, germlining andmanufacturability analysis.

Optimization of Ab1

The Ab1 antibody was selected for further optimization. The affinity forpro/latent-Myostatin was optimized using yeast display. Additionally,the sequence of Ab1 was germlined to reduce the potential immunogenicityliability of non-germline amino acid positions within the human variableregions frameworks.

Affinity Optimization of Ab1 by Yeast Display

The Ab1 parental antibody was optimized for binding topro/latent-Myostatin using an scFv display approach based in yeast.Briefly, three different scFv libraries were created to introduce pointmutations to selected CDR positions based on the amino acid frequencyobserved in natural human antibody repertoires using antibody deepsequencing corresponding to the human frameworks utilized by Ab1. Eachlibrary contained scFv based on the Ab1 sequence with single pointmutations introduced in each CDR such that each variant of the resultingheavy chain or light chain would have three total substitutions, one ineach CDR. The three libraries were used for FACS-based sorting andselection to identify pools of clones with higher binding affinity forpro/latent-Myostatin (FIG. 23). Direct binding of yeast expressed scFvclones was used to select antibodies for conversion to full length IgGexpressed in mammalian cell culture.

Many of the higher affinity scFv clones identified in the yeast campaigncontained a substitution at position 28 of the heavy chain. For someclones, substitution of threonine to asparagine resulted in theincorporation of a non-canonical N-glycosylation motif within CDRH1. AsN-glycosylation within the variable region of an antibody may beundesirable, any clone which contained a glycosylation motif was furthersubstituted to contain alanine at this position.

The binding kinetics to pro- and latent-Myostatin were then assessed byoctet for each of the affinity optimized constructs and compared to thatof the parental Ab1 (discussed in Example 2). All of the clones showedsignificantly increased binding affinity for Myostatin, and two, Ab3 andAb5, were selected based on the selective binding profile over GDF11.

Primary Sequence and Backbone of Anti-Pro/Latent-Myostatin Antibodies

The sequence alignment of the variable regions of parental Ab1 with itsaffinity optimized variants is shown below. Complementarity-determiningregions (CDRs) are defined using the Kabat (underlined) and IMGTnomenclature (bold). Substitutions from parental Ab1 are shown in lowercase text (below and FIG. 24A-24B).

A. Heavy Chain Variable RegionFRAMEWORK 1                  CDR1  FRAMEWORK 2 Ab1 parentalQIQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVA Ab3QIQLVQSGGGVVQPGRSLRLSCAASGFaFSSYGMHWVRQAPGKGLEWVA Ab5QIQLVQSGGGVVQPGRSLRLSCAASGFaFSSYGMHWVRQAPGKGLEWVACDR2             FRAMEWORK 3 Ab1 parentalVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR Ab3VISYDGSiKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR Ab5VISYDGnNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARCDR3             FRAMEWORK 4 Ab1 parentalDLLVRFLEWSHYYGMDVWGQGTTVTVSS (SEQ ID NO: 24) Ab3DLLVRFLEWSHkYGMDVWGQGTTVTVSS (SEQ ID NO: 26) Ab5DLLVRFLEWSHkYGMDVWGQGTTVTVSS (SEQ ID NO: 28)B. Light Chain Variable Region FRAMEWORK 1           CDR1         FRW2Ab1 parental QPVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVHWYQQLPGTAPKLLIY Ab3QPVLTQPPSASGTPGQRVTISCSGStSNIGSNTVHWYQQLPGTAPKLLIY Ab5QPVLTQPPSASGTPGQRVTISCSGSSSNIGgNTVHWYQQLPGTAPKLLIY CDR2   FRAMEWORK 3Ab1 parental SDNQRPSGVPDRFSGSKSGTSASLVISGLQSDDEADYYC Ab3SDdQRPSGVPDRFSGSKSGTSASLVISGLQSDDEADYYC Ab5SDdQRPSGVPDRFSGSKSGTSASLVISGLQSDDEADYYC CDR3      FRAMEWORK 4Ab1 parental AAWDDSLNGVFGGGTKLTVL (SEQ ID NO: 30) Ab3AAWDeSLNGVFGGGTKLTVL (SEQ ID NO: 32) Ab5AAWDeSLNGVFGGGTKLTVL (SEQ ID NO: 34)

Antibody Engineering and Rationale for Isotype Selection

In some embodiments, an antibody useful for myostatin blockade will lackeffector function. Thus for the humanized construct, an IgG4-Fc regionwas selected. Antibodies of the IgG4 isotype poorly bind complement C1qand therefore do not significantly activate complement. These antibodiesalso bind weakly to Fcγ receptors, leading to inefficient or absentantibody-dependent cell-mediated cytotoxicity (ADCC).

To avoid potential complication due to Fab-arm exchange, which is knownto occur with native IgG4 mAbs, Ab1 and its variants were engineeredwith the stabilizing ‘Adair’ mutation (Angal, 1993), where serine 228(EU numbering; residue 241 Kabat numbering) is converted to prolineresulting in an IgG1-like (CPPCP (SEQ ID NO: 58)) hinge sequence. Thisengineered Fc-sequence is used in the production of the approvedantibodies Keytruda, Mylotarg and Tysabri, as well as in a number ofcurrent late-stage clinical candidate mAbs.

Germlining and Immunogenicity Risk Assessment

The Ab1 parental antibody and its variants are fully human IgG4 (S228P),lambda antibodies derived from phage display. The Fc portion of theantibody contains a single stabilizing mutation to prevent Fab armexchange (described above). The IgG4 Fc is not expected to havemeasurable binding to Fc gamma receptors (see Example 2).

The variable framework regions of Ab1 as isolated from the fully humannaïve phage library contains five non-germline amino acids (see belowand FIG. 22). Complementarity determining regions (CDRs) are definedusing the Kabat nomenclature and are underlined. Non-germline residuesare shown in lower case.

A. Heavy Chain Variable Region <-------------FR1------------><CDR><-----FR2----><-----CDR2------> Ab1QIQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGIgHV3-30.v...e............................................................    <--------------FR3-------------><------CDR3-----><---FR4---> Ab1    RFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLLVRFLEWSHYYGMDVWGQGTTVTVSS(SEQ ID NO: 24) IgHV3-30    ..................................... (SEQ ID NO: 36) JH6                                              .................(SEQ ID NO: 59)B. Light Chain Variable Region <FR1 ><CDR1 ><FR2 ><CDR2->  <---------FR1--------><----CDR1---><-----FR2-----><CDR2-> Ab1QPVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVHWYQQLPGTAPKLLIYSDNQRPS IgLV1-44.s................................n................n..... <--------------FR3-------------><--CDR3--><---FR4--> Ab1GVPDRFSGSKSGTSASLVISGLQSDDEADYYCAAWDDSLNGVFGGGTKLTVL (SEQ ID NO: 30)IgLV1-44 .................a......e....... (SEQ ID NO: 60) JL1/2/3                                        ........... (SEQ ID NO: 61)

To mitigate the potential for immunogenicity, additional variants of Ab1molecules were created which substitute the non-germline frameworkresidues to their corresponding germline amino acids. In someembodiments, the substitution pertaining to Ab1 may be similarly appliedto Ab3 and Ab4, or any antibody disclosed herein for which germlining isappropriate.

A sequence alignment of variable regions of Ab1 with its affinityoptimized variants is shown below. A.) heavy chain, B.) light chain.Complementarity determining regions (CDRs) are defined using the Kabat(underlined) and IMGT nomenclature (bold). Framework regionssubstitutions present in parental Ab1 are shown in lower case.

A. Heavy Chain Variable RegionFRAMEWORK 1              CDR1      FRAMEWORK 2 IgHV3-30QVQLVESGGGVVQPGRSLRLSCAASGFTF SSYGMH WVRQAPGKGLEWVA Ab1QiQLVqSGGGVVQPGRSLRLSCAASGFTF SSYGMH WVRQAPGKGLEWVA Ab2QVQLVESGGGVVQPGRSLRLSCAASGFTF SSYGMH WVRQAPGKGLEWVA Ab4QVQLVESGGGVVQPGRSLRLSCAASGFAF SSYGMH WVRQAPGKGLEWVA Ab6QVQLVESGGGVVQPGRSLRLSCAASGFAF SSYGMH WVRQAPGKGLEWVACDR2             FRAMEWORK 3 IgHV3-30VISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR Ab1VISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR Ab2VISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR Ab4VISYDGSIKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR Ab6VISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARCDR3             FRAMEWORK 4 IgHV3-30---------------------------- (SEQ ID NO: 36) Ab1 DLLVRFLEWSHYYGMDVWGQGTTVTVSS (SEQ ID NO: 24) Ab2 DLLVRFLEWSHYYGMDVWGQGTTVTVSS (SEQ ID NO: 25) Ab4 DLLVRFLEWSHKYGMDVWGQGTTVTVSS (SEQ ID NO: 27) Ab6 DLLVRFLEWSHKYGMDVWGQGTTVTVSS (SEQ ID NO: 29) B. Light Chain Variable RegionFRAMEWORK 1           CDR1         FRW2 IgLV1-44QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIY Ab1Q2VLTQPPSASGTPGQRVTISCSGSSSNIGSNTVHWYQQLPGTAPKLLIY Ab2QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVHWYQQLPGTAPKLLIY Ab4QSVLTQPPSASGTPGQRVTISCSGSTSNIGSNTVHWYQQLPGTAPKLLIY Ab6QSVLTQPPSASGTPGQRVTISCSGSSSNIGGNTVHWYQQLPGTAPKLLIY CDR2   FRAMEWORK 3IgLV1-44 SNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYC Ab1SDNQRPSGVPDRFSGSKSGTSASLW_SGLQSoADEADYYC Ab2SDNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYC Ab4SDDQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYC Ab6SDDQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYC CDR3      FRAMEWORK 4 IgLV1-44-------------------- (SEQ ID NO: 60) Ab1 AAWDDSLNGVFGGGTKLTVL (SEQ ID NO: 30) Ab2 AAWDDSLNGV FGGGTKLTVL (SEQ ID NO: 31) Ab4AAWDESLNGV FGGGTKLTVL (SEQ ID NO: 33) Ab6 AAWDESLNGVFGGGTKLTVL (SEQ ID NO: 35)

Three of the five substitutions were found to be away from CDR regionsand therefore have no impact on binding. A Proline at position 2 of thelight chain packs against CDRL3 and substitution to germline Serineactually improves binding to pro/latent-Myostatin by stabilizing the CDRconformation.

The overall antibody is greater than 99% human (calculated as 100% minusthe % non-germline AA excluding CDRH3). There are no chemicalconjugations. The heavy chain CDRH2 sequence contains a potentialisomerization liability (Asp-Gly) which is also present in the germlineIgHV3-30 sequence.

Example 2: Pharmacological Characterization In Vitro PharmacologicalAssays

A total of 24 optimized Ab1 variants were expressed and purified as IgG4and assayed for improved binding and functional activity. The changes tothese molecules included germlining mutations to the parental variableregion, along with mutations in the CDRs which conferred increasedbinding to pro/latent-Myostatin in the affinity maturation screen (seeExample 1).

The Ab1 variants were screened in several different ELISA-based assays,in which the binding to the proMyostatin and latent Myostatin proteins(human, murine, and cynomolgus) was re-assessed, along with a largescreen of negative control proteins to verify that non-specific bindingwas not introduced as a result of the affinity maturation. Negativecontrols included GDF11 proteins (proGDF11, latent GDF11 and matureGDF11), TGFβ proteins, and Activin proteins (proActivin). Additionally,the antibodies were assessed for polyspecificity (which can lead torapid clearance) in a screen similar to that published previously(Hotzel et al., 2012). Any antibodies with significant interactions tonegative control proteins, or with baculovirus particles in thepolyspecificity screen were not considered further as candidates for adevelopment program.

The 24 optimized variants of Ab1 were also assessed in the proMyostatinactivation assay to determine their functional efficacy, and EC50 valuesfrom dose response curves were compared to the parental Ab1 antibody.Most antibodies had equivalent or improved EC50 values, with a fewdisplaying reduced efficacy in this assay. Those with reduced efficacyin the activity assay were excluded from further analysis.

Three variants of Ab1 with improved binding to pro- and latent myostatinwhile retaining specificity for pro- and latent myostatin wereidentified. Binding and activity data for these three variants and theparental Ab1 molecule are summarized in Tables 4-7, sequences are shownin Example 1.

TABLE 4 Binding characteristics of antibodies to human/cynomolgus/mouseproMyostatin to parental Ab1 IgG4. Ab1 Activity Assay - 293T cellsKinetics Analysis - Fortebio Octet EC50 (μM) kon(1/Ms) kdis(1/s) Kd (M)Human 0.274 4.18E+05 1.99E−03 4.76E−09 Cynomolgus 0.5842 3.05E+051.75E−03 5.75E−09 Mouse 0.8386 2.37E+05 2.62E−03 1.10E−08

TABLE 5 Binding characteristics of antibodies to human/cynomolgus/mouseproMyostatin to Ab1 IgG4 with the correct germline residues replaced(Ab2) for non-germlined residues. Ab2 Activity Assay - 293T cellsKinetics Analysis - Fortebio Octet EC50 (μM) kon(1/Ms) kdis(1/s) Kd (M)Human 0.248 4.57E+05 1.56E−03 3.42E−09 Cynomolgus 0.6168 2.78E+051.41E−03 5.08E−09 Mouse 0.7138 2.35E+05 1.97E−03 8.39E−09

TABLE 6 Binding characteristics of antibodies to human/cynomolgus/mouseproMyostatin to Ab3 IgG4 containing the corrected germline residues(Ab4). Ab4 Activity Assay - 293T cells Kinetics Analysis - FortebioOctet EC50 (μM) kon(1/Ms) kdis(1/s) Kd (M) Human 0.179 4.98E+05 2.35E−044.72E−10 Cynomolgus 0.4451 3.01E+05 2.34E−04 7.76E−10 Mouse 0.44662.53E+05 2.72E−04 1.08E−09

TABLE 7 Binding characteristics of antibodies to human/cynomolgus/mouseproMyostatin to Ab5 IgG4 containing the corrected germline residues(Ab6). Ab6 Activity Assay - 293T cells Kinetics Analysis - FortebioOctet EC50 (nM) kon(1/Ms) kdis(1/s) Kd (M) Human 0.151 5.27E+05 2.51E−044.77E−10 Cynomolgus 0.4037 3.50E+05 2.57E−04 7.35E−10 Mouse 0.30682.94E+05 2.81E−04 9.54E−10

Cell-Based, Ex Vivo and In Vivo Biological Activity Assays

Ab1 optimized variants were assessed in the GDF8 activation assay in adose response study. In these experiments, 0.5 μM proMyostatin waspre-incubated with increasing amounts of the test article. Followingthis pre-incubation step, conditioned media from HEK293 cellsoverexpressing the mTll2 and Furin proteases was added to release themature growth factor from proMyostatin. Following incubation at 30° C.overnight, the material was added to 293T cells carrying a SMAD-basedluciferase reporter plasmid, and the activity of the released materialwas recorded. Data from a screen are shown in FIG. 4.

Selectivity for Myostatin Over Other TGFβ Family Members

The selectivity of the candidate antibodies were also assessed by bothbinding and functional assays to verify the lack of cross reactivity toother members of the TGFβ family. Human Myostatin and GDF11 share 90%identity in the mature growth factor domain, and 47% identity in theprodomain regions. From the epitope mapping studies, it was determinedthat the parental Ab1 molecule recognizes an epitope on the prodomain ofproMyostatin and latent myostatin because ELISA assays have shownbinding of this antibody to a construct consisting of the prodomain ofmyostatin. Even though the prodomains of myostatin and GDF11 share lessthan 50% identity, and we do not expect significant cross reactivity,the specificity of the lead antibodies was carefully assessed.

A sensitive assay for detecting interactions between the antibodies ofinterest and negative control reagents was developed. In this assay,biotinylated proGDF11 or biotinylated proMyostatin was immobilized on aForteBio BLI streptavidin-coated sensor tip, which was applied to wellscontaining 30 μg/mL of antibody. Interactions of the analyte with theprotein immobilized on the chip are measured by the magnitude of theresponse of the biosensor chip. The biosensor response after 5 minutesof association (a saturating signal for proGDF8) was compared betweenthe two antigens, and expressed as the percent response for GDF8binding. All antibodies had minimal interactions with proGDF11, comparedto the robust binding events measured for proMyostatin.

TABLE 8 Interactions with proGDF11 at high concentrations of thecandidate molecules GDF11 response, expressed as a percentage of GDF8response Ab1 1.33% Ab2 0.81% Ab4 2.51% Ab6 2.07%

The antibody candidates were also evaluated in a GDF11 activation assay.In this assay, 50 nM proGDF11 is preincubated with increasingconcentrations of the antibody. Following preincubation, conditionedmedium from HEK293 cells overexpressing BMP-1 (a tolloid familyprotease) and PCSK5 (a furin family member specific for GDF11) was addedto proteolytically activate proGDF11. Following overnight incubation at30° C., the reaction mixtures are assessed for GDF11 activity in aSMAD-based reporter cell line. As is shown in Table 8, theanti-myostatin antibodies do not inhibit proGDF11 activation, while apositive control antibody imparts dose-dependent inhibition of GDF11activation.

Binding affinities of antibody candidates were determined using theFortéBio Octet QKe dip and read label free assay system utilizingbio-layer interferometry. Antigens were immobilized to biosensors(streptavidin-coated biosensors for proGDF8, proGDF11 and proActivin;direct amine coupling for all others) in each experiment and theantibodies/constructs were presented in solution at high concentration(50 μg/mL) to measure binding interactions.

Binding affinities of antibodies were determined using the FortéBioOctet QKe dip and read label free assay system utilizing bio-layerinterferometry. Human proGDF8, latent GDF8, proGDF11 and proActivin werebiotinylated and immobilized to streptavidin-coated biosensors(FortéBio). Mature growth factors were immobilized via direct aminecoupling to amine reactive tips according to manufacturer's instructions(FortéBio). In each experiment, the antibodies/constructs were presentedin solution at a single high concentration (50 μg/mL) to measure bindinginteractions. Growth factors were purchased from R&D systems andbiotinylated proteins were produced as described.

TABLE 9a Comparison of antibodies for binding to different forms ofseveral TGFβ family members. Ab2 Ab4 Ab6 Ab1 Pro GDF8 7.35E−09 9.24E−108.89E−10 6.23E−09 Latent GDF8 7.84E−09 1.10E−09 1.12E−09 9.06E−09 MatureGDF8 − − − − Pro GDF11 − *1.25E−07  *6.07E−08  − Mature GDF11 − − − ProActivin A − − − − Mature Activin A − − − − BMP 9 − − − − BMP10 − − − −Mature TGFB1 − − − − *Non-specific binding.

Results from the antigen binding study are summarized in Table 9a.Experiments with no detectable binding are noted by a minus sign (−).There are some calculated Kd values that were fitted to data with poorbinding response, which is indicated in the table as weak non-specificbinding (*).

As the proGDF8 sample used in Table 9a contained approximately 10-15%latent GDF8, a separate experiment was used to confirm proGDF8 bindingto human and murine GDF8 antigens specifically. In addition, primedGDF8, in which proGDF8 is proteolytically cleaved by both a proproteinconvertase and tolloid protease was also assessed for binding affinityto Ab2 and AbMyo. For these experiments, a homogenous preparation ofhuman proGDF8 was purified from stably integrated 293 cells cultured inthe presence of 30 μM decanoyl-RVKR-CMV. Primed human GDF8 was producedby in-vitro cleavage of proGDF8 utilizing conditioned media frommTll2-overexpressing cells and purified Furin protease. In the bindingexperiments with these proteins, 150 nM of Ab2 or AbMyo was used tosaturate the immobilization sites on human Fc capture tips (FortéBio),and the association and dissociation of 150 nM analyte was evaluated.

Analysis of binding affinities to murine proteins were also assessed andare reported in Table 9b. Murine proGDF8 protein was produced byremoving all mature and latent murine GDF8 from the sample via negativeselection with an antibody that tightly recognizes latent and matureGDF8 (AbMyo2). 50 nM of antibody was used to saturate an anti-human Fccapture tip (FortéBio). Initially, all antibodies were tested against asingle 200 nM concentration of murine proGDF8, murine latent GDF8, andmature GDF8. If binding was observed, a Kd value was determined byimmobilizing the antibody as previously described and using analyte intitration from 200 to 0.82 nM by 3-fold dilutions. The Kd was determinedusing a global fit with FortéBio data analysis software 8.2. For bindingto mature myostatin, 5 ug/mL of growth factor (R&D systems) was coupledto amine reactive sensor tips (FortéBio) in acetate buffer at pH 5. Allantibodies were initially tested at 333 nM for binding to thismyostatin-coupled sensor. Antibodies that showed binding were thentested in concentrations ranging from 333 to 1.37 nM by 3 folddilutions. A global fit was used to determine the Kd of the interactionusing FortéBio data analysis 8.2.

TABLE 9b Comparison of antibodies for binding to different forms ofhuman and murine GDF8. Ab2 AbMyo Human Pro GDF8 2.9E−09 — Human latentGDF8 2.4E−9  3.87E−10 Human primed GDF8 8.66E−09  8.83E−10 Mature GDF8 — 4.7E−11 Murine ProGDF8 2.3E−09 — Murine Latent GDF8 2.0E−09  <1E−12

Results from an antigen binding study are summarized in Table 9b.Experiments with no detectable binding are noted by a minus sign (−).Some values, labeled as <1 E-12, had very slow dissociation rates makingthe high affinity unable to be quantified. Surprisingly, AbMyo wasunable to recognize recombinant proGDF8, which is different than resultsreported in Latres et al 2015, in which the authors reported associationof AbMyo with proGDF8 in an immunoprecipitation experiment from serum ofmice that were dosed with the antibody which could produce artifacts.Another surprising result is the interaction between Ab2 and primedGDF8, a complex of GDF8 with tolloid-cleaved prodomain. This result isunexpected because Ab2 blocks tolloid cleavage of the prodomain andsuggests that the interaction of Ab2 with proGDF8 and latent GDF8 doesnot require an intact tolloid cleavage site.

Evaluation of Fc-Region Functionality

In some embodiments, anti-pro/latent-Myostatin therapy involves bindingto a soluble target (pro/latent-Myostatin) and preventing proteolyticactivation. In some embodiments, antibody dependent cell-mediatedcytotoxicity and complement fixation are not involved in this process.Ab1 and its related variants were engineered to contain an IgG4-Fcregion. It is understood that IgG4 antibodies generally lack effectorfunction due to their weak binding to complement component C1q and Fcγreceptors.

To demonstrate the reduced capacity for effector function, Ab1 andrelated antibodies were tested for binding to CD64 (FcgRI) and C1q byELISA. For comparison, an IgG1 variant of Ab1 was also prepared. In thisassay, all IgG4 antibodies showed significantly weaker binding (10 to20-fold) to CD64 and C1q compared to IgG1. The relative binding valuesat the EC50 are listed in Table 10.

TABLE 10 Relative binding affinities of Ab2 and related antibodies toCD64. Relative CD64 Relative C1q Binding @ Binding @ Antibody IsotypeEC50(%) EC50(%) Ab1-G1 IgG1 100 100  Ab1 IgG4 (S228P) 10 ND Ab2 IgG4(S228P) 5 8 Ab3 IgG4 (S228P) 5 5 Ab5 IgG4 (S228P) 8 9 Not determined

The apparent binding affinities of Ab1 and its related variants to CD64and C1q are similar to other IgG4 clinical candidate antibodies, and areconsiderably reduced compared to antibodies of the IgG1 isotype. Basedon the biology of IgG4 antibodies, it is therefore concluded that theanti-pro/latent-Myostatin antibodies will not induce appreciableeffector function in vivo.

Efficacy in Animal Models

Based on in vitro characterization, four antibodies were chosen to testin an in vivo study (Ab7, Ab1, Ab8 and Ab9). The objective of the studywas to assess the ability of these four candidate antibodies to modulatemuscle mass mice. Five (5) groups of ten (10) female SCID mice receivedtest article administration by intraperitoneal (IP) injection once perweek on Days 0, 7, 14, 21, 28, and 35. Prior to test articleadministration (Day 0), all animals underwent grip strength evaluation.Grip strength evaluation was also performed on the last day of the study(Day 42). On Day 0, blood was collected via retro-orbital bleed forassessment of complete blood counts (CBC). Following dosing, animalswere evaluated daily for body weight and general health observations. OnDay 42, following grip strength assessment, animals were sacrificed viaCO₂ overdose and blood was collected via cardiac puncture for CBCassessment. Additional blood was collected for the preparation ofplasma. Various tissues were isolated and weighed. The muscles collectedwere: gastrocnemius, pectoralis, soleus, triceps, tibialis anterior,quadriceps (rectus femoris) and diaphragm. The organs collected were:heart, kidney, spleen, liver and inguinal white adipose tissue. Alltissues were weighed and snap frozen except for the gastrocnemiusmuscles which were fixed in formalin (leg 1) and OCT (leg 2) forhistologic analysis.

Summary

The mean daily percent weight change data for animals in study SCH-02are shown in FIG. 6. Animals in all five groups gained weight on aweekly basis. Animals treated with the antibody Ab1 had the largestincrease in body weight (14.6%) as depicted in FIG. 6. Only animalstreated with Ab1 had a statistically significant increase in meanpercent body weight change in comparison to animals in the vehicle (PBS)control group (FIG. 6).

The weights for the dissected muscles are plotted in FIGS. 7 and 8.Animals treated with Ab1 had statistically significant increases ingastrocnemius (FIG. 7A) and diaphragm (FIG. 8B) weights compared tovehicle (PBS) control treated animals, 27.6% and 49.8%, respectively.Additional muscles from Ab1 treated animals showed increases in weightcompared to the PBS control, but these differences were notstatistically significant. There were no statistically significantdifferences between treatment groups for the mean tissue weights ofheart, spleen, kidney, liver, and adipose tissue.

SCID Dose Response Study

In the in vivo study (above) animals dosed with Ab1 at 25 mg/kg onceweekly for 6 weeks showed statistically significant increases in bodyweight and muscle weights (gastrocnemius and diaphragm) compared toanimals dosed with the vehicle (PBS). This muscle enhancing activity ofAb1 was next investigated in more detail in a dose response study inSCID mice. In this study, whether the magnitude of the effect on musclemass could be increased by increasing the dose of Ab1 to as high as 60mg/kg/wk and whether the magnitude of the effect on muscle mass could bedecreased by decreasing the dose of Ab1 to as low as 2 mg/kg/wk wereexamined. In this study, the activity of Ab1 was compared to two moreantibodies (Ab8, which was originally tested in the study describedabove Ab10).

Ten (10) groups of ten (10) female SCID mice received test articleadministration by intraperitoneal (IP) injection (10 ml/kg) twice perweek on Days 0, 3, 7, 10, 14, 17, 21, and 24. The doses of test articleswere as follows: Ab1 (30 mg/kg, 10 mg/kg, 3 mg/kg and 1 mg/kg), Ab10 (10mg/kg and 3 mg/kg), and Ab8 (10 mg/kg and 3 mg/kg). Control groups weredosed with PBS and IgG-control (30 mg/kg). Treatment groups aredescribed in Table 11. Animals were 10 weeks old at the start of thestudy. Body weight was measured on day −4 and twice per week throughoutthe study, corresponding with dosing days. Body mass compositionparameters (fat mass, lean mass and water content) were measured by EchoMRI (QNMR) on days −4, 7, 14, 21 and 28. Thirty (30) days after thefirst dose of antibody, animals were sacrificed via CO₂ overdose andblood was collected via cardiac puncture for CBC assessment and plasmapreparation. Additionally, upon study termination, various tissues wereisolated and weighed. The muscles collected were: gastrocnemius, soleus,tibialis anterior, quadriceps (rectus femoris) and diaphragm. Muscleswere dissected from both the right and left legs of study mice. Foranalysis the weights of the individual muscles from both legs werecombined and the average muscle weight in grams was calculated. Theother tissues collected were: heart, kidney, spleen, liver and adiposetissue. All tissues were weighed and then snap frozen except for thegastrocnemius muscles which were fixed in formalin (left leg) and OCT(right leg) for histologic analysis.

TABLE 11 Study design Treat- # doses ment per Total dose Animal GroupTest Article dose week per week IDs 1 PBS Control 2 0  1-10 2 IgGControl (30 mg/kg) 2 60 mg/kg/wk 11-20 3 Ab1 (30 mg/kg) 2 60 mg/kg/wk21-30 4 Ab1 (10 mg/kg) 2 20 mg/kg/wk 31-40 5 Ab1 (3 mg/kg) 2 6 mg/kg/wk41-50 6 Ab1 (1 mg/kg) 2 2 mg/kg/wk 51-60 7 Ab10 (10 mg/kg) 2 20 mg/kg/wk61-70 8 Ab10 (3 mg/kg) 2 6 mg/kg/wk 71-80 9 Ab8 (10 mg/kg) 2 20 mg/kg/wk81-90 10 Ab8 (3 mg/kg) 2 6 mg/kg/wk  91-100

Mean percent weight change and mean percent lean mass change data foranimals treated with vehicle (PBS), IgG control and different doses ofAb1 are shown in FIG. 9. Animals treated with Ab1 at 20 and 60 mg/kg/wkdoses had significant increases in body weight on day 28 of the studycompared to IgG control treated animals, 15.3% and 14.4%, respectively(FIG. 9A). All four groups of animals treated with Ab1 (60, 20, 6 and 2mg/kg/wk doses) had statistically significant increases in lean mass onday 28 of the study compared to IgG control treated animals, 14.1%,12.4%, 17.1%, and 15.5%, respectively (FIG. 9B).

The weights for four muscles (quadriceps, gastrocnemius, tibialisanterior and diaphragm) are plotted in FIG. 10. The soleus muscle wasalso dissected, but the small size of the muscle resulted in anextremely variable data set. Animals treated with all doses of Ab1 hadstatistically significant increases in muscle weights compared to IgGcontrol animals (FIG. 10). The mean percent changes in muscle masscompared to IgG control are shown above the corresponding bar on eachmuscle graph. Mean percent weight changes for quadriceps muscle rangedfrom 20.5% for the highest dose to 10.7% for the lowest dose (FIG. 10A).Mean percent weight changes for gastrocnemius muscle ranged from 17.7%for the highest dose to 15.9% for the lowest dose (FIG. 10B). Meanpercent weight changes for tibialis anterior muscle ranged from 24.0%for the highest dose to 18.0% for the lowest dose (FIG. 10C). Meanpercent weight changes for diaphragm muscle were greater than 30% forall dose groups (FIG. 10D). There were no statistically significantdifferences between treatment groups for the mean tissue weights ofheart, spleen, kidney, liver, and adipose tissue.

Ab1 Treatment in a Dexamethasone Induced Muscle Atrophy Model

Given the ability of the anti-myostatin antibody Ab1 to build musclemass in healthy SCID mice, it was determined whether Ab1 treatment couldalso protect animals from treatments that induce muscle atrophy. A modelof corticosteroid-induced muscle atrophy was established by treatinganimals for two weeks with dexamethasone in their drinking water. Thedose chosen (2.5 mg/kg/day) was able to induce significant decreases inlean body mass and the mass of individual hindlimb muscles. In thefollowing experiment, animals were treated with different doses of Ab1to determine if it could protect animals from this dexamethasone-inducedmuscle atrophy.

In this study, eight (8) groups of ten (10) male mice (C57BL/6) wereenrolled in the study at 13.5 weeks of age. Starting on day 0 of thestudy, mice were given either normal drinking water (groups 1-4) orwater containing dexamethasone (groups 5-8). Test articles wereadministered by intraperitoneal (IP) injection (10 ml/kg) twice per weekon Days 0, 3, 7, and 10. The test articles and doses were as follows:PBS (groups 1 and 5), 10 mg/kg IgG Ctl (groups 2 and 6), 10 mg/kg Ab1(groups 3 and 7), and 1 mg/kg Ab1 (groups 4 and 8). Treatment groups aredescribed in Table 12. Body weight was measured at least twice per weekthroughout the study. Body mass composition parameters (fat mass, leanmass and water content) were measured by Echo MRI (QNMR) on days −1, 6,and 13. Fourteen (14) days after the first dose of antibody, animalswere sacrificed via CO₂ overdose and blood was collected via cardiacpuncture for plasma preparation. Additionally, upon study termination,various tissues were isolated and weighed. The muscles collected were:gastrocnemius, soleus, tibialis anterior, quadriceps (rectus femoris)and diaphragm. Muscles were dissected from both the right and left legsof study mice. For analysis the weights of the individual muscles fromboth legs were combined and the average muscle weight in grams wascalculated. The other tissues collected were: heart, kidney, spleen,liver and adipose tissue. All tissues were weighed and then snap frozenexcept for the gastrocnemius muscles which were fixed in formalin (leftleg) and OCT (right leg) for histologic analysis.

TABLE 12 Treatment groups for Dexamethazone-induced atrophy model studyTreatment Dexamethasone # doses Total dose Animal Group in drinkingwater Test Article dose per week per week IDs 1 none PBS Control 2 0 1-10 2 none IgG Control (10 mg/kg) 2 20 mg/kg/wk 11-20 3 none Ab1 (10mg/kg) 2 20 mg/kg/wk 21-30 4 none Ab1 (1 mg/kg) 2 2 mg/kg/wk 31-40 5 2.5mg/kg/day PBS Control 2 0 51-60 6 2.5 mg/kg/day IgG Control (10 mg/kg) 220 mg/kg/wk 61-70 7 2.5 mg/kg/day Ab1 (10 mg/kg) 2 20 mg/kg/wk 71-80 82.5 mg/kg/day Ab1 (1 mg/kg) 2 2 mg/kg/wk 81-90

In this experiment it was determined whether treatment of mice with theanti-myostatin antibody Ab1 could protect animals from corticosteroidinduced muscle atrophy. During the study body weight was measured twiceweekly and lean mass by QNMR on days −1, 6 and 13. The mean percentweight change and mean percent lean mass change data for animals in thenon-diseased control group (group 1) and the dexamethasone treatedgroups (groups 5-8) are shown in FIG. 11. There were no significantdifferences in mean percent body weight change between any of thesetreatment groups on day 14 (FIG. 11A). Treatment of mice withdexamethasone for two weeks led to a significant decrease in lean bodymass (groups 5 and 6) compared to a control group (group 1) that wasgiven normal drinking water (FIG. 11B). However, mice treated with bothdexamethasone and the antibody Ab1 at 20 mg/kg/wk (group 7) showed nosignificant difference in percent change in lean body mass on day 14 ofthe study compared to the control group (group 1). Animals treated withAb1 at 20 mg/kg/wk, but not 2 mg/kg/wk, showed a significant differencein percent change in lean body mass on day 14, when compared to eitherof the dexamethasone treated control groups (groups 5 and 6).

At the end of the two week treatment with dexamethasone and the testarticles, individual muscles were dissected and weighed. The weight datafor two muscles (gastrocnemius and quadriceps) are plotted in FIGS.12A-12B. Animals that received dexamethasone via their drinking waterand also received either PBS or IgG Control antibody showed significantatrophy in gastrocnemius and quadriceps muscles (groups 5 and 6)compared to the non-diseased control group (group 1). Animals treatedwith both dexamethasone and Ab1 at 20 mg/kg/wk (group 7), but not 2mg/kg/wk, showed a significant difference in muscle weights whencompared to either of the dexamethasone treated control groups (groups 5and 6). In addition, mice treated with both dexamethasone and theantibody Ab1 at 20 mg/kg/wk (group 7) showed no significant differencein gastrocnemius and quadriceps weights when compared to thenon-diseased control group (group 1). The mean percent difference inmuscle weight of each group compared to the mean muscle weight of thecontrol group (group 1, PBS and water) is shown in FIGS. 12C-12D. Thepercent decreases in gastrocnemius mass induced by dexamethasonetreatment in the PBS and IgG Ctl groups were 16.5% and 18.9%,respectively. In contrast, animals treated with both dexamethasone and20 mg/kg/wk of Ab1 only had a 4.0% decrease in gastrocnemius muscle masswhich was not statistically different from the non-diseased controlgroup (group 1). While animals treated with both dexamethasone and 2mg/kg/wk Ab1 (group 8) only had a 10% decrease in gastrocnemius musclemass, the muscle mass decrease for this group was not statisticallydifferent than the decreases for the PBS and IgG control groups (groups5 and 6). Similar results were seen for the quadriceps muscle (FIG.12D).

Ab1 Treatment in a Casting Induced Muscle Atrophy Model

Given the ability of the anti-myostatin antibody Ab1 to build musclemass in healthy SCID mice, whether Ab1 treatment could also protectanimals from treatments that induce muscle atrophy was investigated. Amodel of disuse atrophy was established by casting the right leg of micefor two weeks. Casting the right leg with the foot in a plantar flexionposition for this time period was able to induce significant decreasesin the mass of individual hindlimb muscles. In the following experimentanimals were treated with different doses of Ab1 to determine the extentto which it protects animals from this casting induced muscle atrophy.

TABLE 13 Treatment groups for casting-induced atrophy model studyTreatment # doses Total dose Animal Group Casting Test Article dose perweek per week IDs 1 No cast PBS Control 2 0  1-10 2 No cast IgG Control(10 mg/kg) 2 20 mg/kg/wk 11-20 3 No cast Ab1 (10 mg/kg) 2 20 mg/kg/wk21-30 4 No Cast Ab1 (1 mg/kg) 2 2 mg/kg/wk 31-40 5 Casted PBS Control 20 51-60 6 Casted IgG Control (10 mg/kg) 2 20 mg/kg/wk 61-70 7 Casted Ab1(10 mg/kg) 2 20 mg/kg/wk 71-80 8 Casted Ab1 (1 mg/kg) 2 2 mg/kg/wk 81-90

In this study, eight (8) groups of ten (10) male mice (C57BL/6) wereenrolled in the study at 14.5 weeks of age. Starting on day 0 of thestudy, mice were placed under anesthesia and a cast was applied to theright hindlimb with the foot in a plantar flexion position (groups 5-8).The control groups (groups 1-4) were also placed under anesthesia but nocast was placed on the hindlimb. Test articles were administered byintraperitoneal (IP) injection (10 ml/kg) twice per week on Days 0, 3,7, and 10. The test articles and doses were as follows: PBS (groups 1and 5), 10 mg/kg IgG Ctl (groups 2 and 6), 10 mg/kg Ab1 (groups 3 and7), and 1 mg/kg Ab1 (groups 4 and 8). Treatment groups are described inTable 13. Body weight was measured at least twice per week throughoutthe study. Body mass composition parameters (fat mass, lean mass andwater content) were measured by Echo MRI (QNMR) on days −1, 7, and 14.Fourteen (14) days after the first dose of antibody, animals weresacrificed via CO₂ overdose and blood was collected via cardiac puncturefor plasma preparation.

Additionally, various tissues were isolated and weighed. The musclescollected were: gastrocnemius, soleus, plantaris, tibialis anterior, andquadriceps (rectus femoris). For analysis the weights of the individualmuscles from the right hindlimb of the animals were collected. The othertissues collected were: heart, adipose and spleen. All tissues wereweighed and then snap frozen except for the gastrocnemius muscles whichwere fixed in formalin for histologic analysis.

Summary

In this experiment, whether treatment of mice with the anti-myostatinantibody Ab1 could protect mice from disuse muscle atrophy induced bycasting of the right hindlimb was tested. During the study, body weightwas measured twice weekly and lean mass was measured by QNMR on days −1,7 and 14. The mean percent weight change and mean percent lean masschange data for animals in the non-diseased control group (group 1) andthe groups that were casted for two weeks (groups 5-8) are shown in FIG.13. Casting of the right hind limb did not have any negative effects onbody weight gain (FIG. 13A) and any differences in lean mass of groupswere not significant (FIG. 13B).

At the end of the two week study individual muscles were dissected andweighed. The weight data for two muscles (gastrocnemius and quadriceps)are plotted in FIGS. 14A-14B). Animals that had their leg casted andalso received either PBS or IgG Control antibody showed significantatrophy in gastrocnemius and quadriceps muscles (groups 5 and 6)compared to the non-casted control group (group 1). Animals that wereboth casted and dosed with Ab1 at 20 mg/kg/wk (group 7), but not 2mg/kg/wk, showed a significant difference in muscle weights whencompared to either of the casted control groups (groups 5 and 6). Inaddition, casted mice that were treated with the antibody Ab1 at 20mg/kg/wk (group 7) showed no significant difference in gastrocnemius andquadriceps weights when compared to the non-casted control group (group1). The mean percent difference in muscle weight of each group comparedto the mean muscle weight of the non-casted control group (group 1) isshown in FIGS. 14C-14D. The percent decreases in gastrocnemius massinduced by casting in the PBS and IgG Ctl groups were 22.8% and 23.5%,respectively. In contrast, casted mice that were treated with 20mg/kg/wk of Ab1 only had a 10.0% decrease in gastrocnemius muscle mass.This difference was found to be statistically different from the castedcontrol groups that received PBS and IgG Ctl antibody (group 5 and 6).The muscle mass decrease for the casted mice treated with 2 mg/kg/wk Ab1was not statistically different than the decreases for the PBS and IgGcontrol groups (groups 5 and 6). Similar results were seen for thequadriceps muscle (FIG. 14D).

The domain structure of proMyostatin and latent Myostatin, with proteasecleavage sites indicated, is shown in FIG. 16A. An example of partiallyproprotein convertase cleaved proMyostatin run on an SDS PAGE gel isshown in FIG. 16B. Under reducing conditions, the protein bandsconsisted of the proMyostatin monomer (˜50 kD), prodomain (˜37 kD) andgrowth factor (12.5 kD).

Ab1 binds specifically to proMyostatin and latent Myostatin, with nobinding observed to other members of the TGFβ superfamily, most notablythe corresponding forms of GDF11 (FIG. 17A). Ab1 was administered at ahigh concentration (50 ug/mL) to Forte-Bio BLI tips coated with theindicated antigen and the on and off rates were measured to obtain anapproximate Kd value. The magnitude of biosensor response, indicating abinding event, is graphically represented by black bars, and thecalculated Kd is indicated in orange. Furthermore Ab1 blocks theactivation of proMyostatin, but not proGDF11 (FIG. 17B).

SCID Dose Response Study with Ab1, Ab2, Ab4 and Ab6

The previous in vivo studies with Ab1 have demonstrated that Ab1 canincrease muscle mass in healthy animals as well as prevent muscle lossin mouse models of muscle atrophy (dexamethasone and casting inducedatrophy). Antibody engineering efforts identified three antibodies within vitro characteristics that were better than Ab1. In this study, inSCID mice, the in vivo activity of these antibodies was compared atthree different doses to the already established activity of Ab1.

Fourteen (14) groups of eight (8) female SCID mice received test articleadministration by intraperitoneal (IP) injection (10 ml/kg) twice perweek on Days 0, 3, 7, 10, 14, 17, 21, and 24. The doses of test articleswere as follows: Ab1, Ab2, Ab4 and Ab6 were given at 3 different doses(10 mg/kg, 1 mg/kg, and 0.25 mg/kg) and the IgG-Ctl antibody was givenat 10 mg/kg. Treatment groups are described in Table 14. Animals were 10weeks old at the start of the study. Body weight was measured twice perweek throughout the study, corresponding with dosing days. Body masscomposition parameters (fat mass, lean mass and water content) weremeasured by Echo MRI (QNMR) on days 0, 7, 14, 21 and 28. Twenty-eight(28) days after the first dose of antibody, animals were sacrificed viaCO₂ overdose and blood was collected via cardiac puncture for plasmapreparation.

Additionally, various tissues were isolated and weighed. The musclescollected were: gastrocnemius, soleus, tibialis anterior, quadriceps(rectus femoris), extensor digitorum longus, and diaphragm. Muscles weredissected from both the right and left legs of study mice. For analysisthe weights of the individual muscles from both legs were combined andthe average muscle weight in grams was calculated. The other tissuescollected were: heart, kidney, spleen, liver and adipose tissue. Alltissues were weighed and then snap frozen except for the leftgastrocnemius muscles which was fixed in formalin for histologicanalysis.

TABLE 14 Treatment groups for dose response model study Treat- # dosesment per Total dose Animal Group Test Article dose week per week IDs 1PBS Control 2 0 1-8 2 IgG Control (10 mg/kg) 2 20 mg/kg/wk  9-16 3 Ab1(10 mg/kg) 2 20 mg/kg/wk 17-24 4 Ab1 (1 mg/kg) 2 2 mg/kg/wk 25-32 5 Ab1(0.25 mg/kg) 2 0.5 mg/kg/wk 33-40 6 Ab2 (10 mg/kg) 2 20 mg/kg/wk 41-48 7Ab2 (1 mg/kg) 2 2 mg/kg/wk 49-56 8 Ab2 (0.25 mg/kg) 2 0.5 mg/kg/wk 57-649 Ab4 (10 mg/kg) 2 20 mg/kg/wk 65-72 10 Ab4 (1 mg/kg) 2 2 mg/kg/wk 73-8011 Ab4 (0.25 mg/kg) 2 0.5 mg/kg/wk 81-88 12 Ab6 (10 mg/kg) 2 20 mg/kg/wk89-96 13 Ab6 (1 mg/kg) 2 2 mg/kg/wk  97-104 14 Ab6 (0.25 mg/kg) 2 0.5mg/kg/wk 105-112

Mean percent lean mass changes (from day 0) data for animals treatedwith vehicle (PBS), IgG control, and different doses of Ab1, Ab2, Ab4and Ab6 are shown in FIG. 15. Animals treated with Ab1, Ab2, Ab4, andAb6 at a 20 mg/kg/wk dose level had significant increases in lean masson day 21 and day 28 of the study compared to IgG control and vehicle(PBS) treated animals. Animals treated with Ab1 and Ab2 at a 2 mg/kg/wkdose level also had significant changes in lean mass at day 21 and 28 ofthe study. There were no significant changes in lean mass from thecontrol groups for animals treated with Ab1, Ab2, Ab4 and Ab6 at a 0.5mg/kg/wk dose level.

At the end of the study (day 28) muscles were collected and weighed. Theweights for quadriceps (rectus femoris) and gastrocnemius muscles areplotted in FIGS. 18A and 18B. Animals treated with Ab1, Ab2, Ab4, andAb6 at a 20 mg/kg/wk dose level had significant increases ingastrocnemius and quadriceps (rectus femoris) muscle weights compared tovehicle (PBS) treated animals. Animals treated with Ab2 and Ab4 at a 2mg/kg/wk dose level also had significant changes in gastrocnemius muscleweights. Animals treated with Ab2 at a 2 mg/kg/wk dose level also hadsignificant changes in quadriceps (rectus femoris) muscle weights. Therewere no significant changes in muscle mass from the control groups foranimals treated with Ab1, Ab2, Ab4 and Ab6 at a 0.5 mg/kg/wk dose level.Percent differences in gastrocnemius and quadriceps (rectus femoris)muscle weights (when compared to the vehicle group) of animals treatedwith different doses of Ab1, Ab2, Ab4 and Ab6 are listed in FIG. 18C.

Duration of Action Study with Ab1 in SCID Mice

The ability of Ab1 to increase lean mass in SCID mice after a singledose and after 3 weekly doses was tested. Seven (7) groups of eight (8)female SCID mice received test article administration by intraperitoneal(IP) injection (10 ml/kg) either once at day 0 (groups 1-4) or once perweek on days 0, 7 and 14 (groups 5-7). See Table 15. Antibodies (IgGcontrol, Ab1 and AbMyo) were dosed at 10 mg/kg. Animals were 10 to 11weeks old at the start of the study. Body weight was measured twice perweek throughout the study, corresponding with dosing days. Body masscomposition parameters (fat mass, lean mass and water content) weremeasured by Echo MRI (QNMR) on days 0, 7, 14, and 21.

TABLE 15 Treatment groups and dosing frequency. Treatment Test DoseDosing Group Article (mg/kg) Frequency 1 PBS 0 Once Control 2 IgG 10Once CTL 3 Ab1 10 Once 4 AbMyo 10 Once 5 IgG 10 Once CTL Weekly 6 Ab1 10Once Weekly 7 AbMyo 10 Once Weekly

Mean percent lean mass change data for animals treated with vehicle(PBS), IgG control, Ab1, AbMyo are shown in FIG. 19. The data areexpressed as change in lean mass from day 0 of the study. At 21 daysafter a single dose of test article, animals treated with Ab1 (group 3)had significant increases in lean mass (compared to IgG controlanimals-group 1) that were indistinguishable from the lean mass changesafter 3 doses of Ab1 (group 6). These changes in lean mass were alsocomparable to changes seen in animals treated with a single dose (group4) or with 3 weekly doses (group 7) of AbMyo.

Example 3: Chemistry/Pharmaceutical Sciences

Ab2 is a humanized monoclonal antibody of the IgG4 subtype with Prolinesubstituted for Serine at position 228. This generates an IgG1-likehinge sequence and minimizes the incomplete formation of inter-chaindisulfide bridges which is characteristic of IgG4. The complete aminoacid sequence of the heavy and light chains of Ab2 are shown below. Thecomplementarity-determining regions (CDRs) are underlined. Bolded NSTsequence: N-linked glycosylation consensus sequence site; Bolded DPsequences are potential cleavage sites; Bolded NX sequences, wherein Xcan be S, T, or G are potential deamidation sites; Bolded DX sequences,wherein X can be G, S, T, or SDG are potential isomerization sites;Bolded methionines are potential methionine oxidation sites; Bolded Q isan expected N-terminal pyroglutamic acid (FIGS. 21A-21B).

Molecular modeling of Ab1 identified several potential sites ofpost-translational modifications. Two asparagines in the light chain andseven asparagines in the heavy chain are susceptible to deamidation. Twoof these residues are located within CDR regions of the heavy chain.

Native IgG4 mAbs may have incomplete formation of inter-heavy chaindisulfide bridges, with the two half molecules (each containing oneheavy and one light chain) maintained in the intact antibody structureby noncovalent interactions. IgG4 molecules may be prone to exchange ofhalf-molecules in vitro and in vivo, and the level of half moleculesmust be consistent across manufacturing batches. The substitution of Serto Pro in the backbone of the IgG4 structure results in an IgG1-likehinge sequence, thereby enabling the formation of inter-chain disulfidebonds and markedly stabilizing the antibody structure. The integrity andstability of the hinge region is monitored during development withextended characterization, using such assays as non-reducing capillaryelectrophoresis and quantitation of free sulfhydryls. The potential forchain swapping is monitored in vivo.

Summary

A pro/latent-Myostatin-specific antibody that blocks the activation ofproMyostatin and/or latent myostatin is provided herein. Administrationof this activation-blocking antibody to healthy mice increases lean bodymass and muscle size, with only a single dose needed to sustain themuscle-enhancing effect over a 1 month period. Additionally, antibodyadministration protects healthy mice from muscle atrophy in two separatemodels of muscle wasting. The data demonstrate that blocking myostatinactivation promotes robust muscle growth and prevents muscle atrophy invivo, and represents an alternative mechanism for therapeuticinterventions of muscle wasting.

Example 4: Analysis of Pro- and Latent-Myostatin in Muscle Atrophy

Western blots were performed to determine the presence of pro and latentMyostatin in muscle tissue and in circulation during muscle atrophy aswell as during normal conditions. A standard model of muscle atrophyinvolves treating mice with 2.5 mg/kg/week Dexamethasone (dosed indrinking water) and muscle and plasma were collected after 2 weeks oftreatment. This model regularly leads to 15-25% decrease in muscle massover the course of treatment. Control muscle and plasma were collectedat the same time from mice not treated with Dexamethasone. Rectusfemoris, tibialis anterior, and soleus muscles were dissected out, flashfrozen in liquid nitrogen, and stored at −80 C until ready to use.Muscle lysates were generated by pulverization, followed by lysis inT-PER buffer supplemented with protease and phosphatase inhibitors.Plasma was collected by standard methods and stored at −80 C.

Multiple samples containing equal concentrations of protein wereseparated by PAGE gels and Western blotted onto PVDF membrane. Formuscle lysates, 10-50 ng total protein was loaded onto the gel. Plasmawas diluted 1:10 in PBS and 10 μl of each sample was loaded onto thegel. As size standards, 0.1-1 ng recombinant pro and/or latent Myostatinwere also loaded onto the gel. Identification of Myostatin protein wasaccomplished using an antibody recognizing the prodomain of Myostatin(AF1539, R&D Systems). This analysis shows that proMyostatin is thepredominant form in muscle, while the latent Myostatin is the primaryform in Plasma (FIG. 25). Furthermore, it was demonstrated that, in micewith muscle atrophy induced by Dexamethasone, proMyostatin is increasedin muscle tissue, while latent Myostatin is decreased in plasma.

To confirm these results, the Western blots were repeated usingfluorescent labeling and detection (Azure Biosystems). The relativelevels of each of the Myostatin forms in plasma and in rectus femorisand tibialis anterior muscles from normal and Dexamethasone-treated micewere quantified. These data confirm the results described above, showinga 2- to 2.5-fold increase in proMyostatin in both muscles, and a2.3-fold decrease in latent Myostatin in plasma (FIG. 26).

Based on these data, a model for Myostatin “flux” in normal and diseasedmuscle is given. As demonstrated, in normal muscle (FIG. 28A),proMyostatin is produced in muscle and converted to latent Myostatinthrough cleavage by Furin protease, which may occur either inside oroutside of the cell (Anderson et al., 2008). Some fraction of the latentMyostatin in muscle is then released into the circulation, forming acirculating pool of latent Myostatin. In muscle atrophy, an increase inthe active Myostatin growth factor is produced, driving muscle atrophy.This increase is thought to be caused by the upregulation ofproMyostatin levels in muscle and the increased conversion of latentMyostatin to the active growth factor (FIG. 28B). The data outlined heredirectly support the first step of this model, showing increasedproMyostatin in muscle. The data also support the second step asdecreased muscle mass in Dexamethasone-treated mice was observed,indicating an increased production of mature Myostatin, without aconcomitant increase in latent Myostatin in muscle. Accordingly, thelevel of Myostatin is plasma was decreased, suggesting an increasedconversion to mature Myostatin.

Example 5: Immunoprecipitation from Murine Serum and Muscle Tissue

Immunoprecipitations were performed to determine the presence ofproMyostatin in circulation, and to investigate the binding of Ab2 andAbMyo to endogenous myostatin precursors in serum and muscle. Ab2recognizes the major form of Myostatin in muscle. Results shown in FIG.27 demonstrate that a pool of serum proMyostatin precipitates with Ab2,suggesting that there is extracellular proMyostatin present in vivo. Inaddition to binding to proMyostatin, latent Myostatin, and otherpartially processed forms of myostatin in serum, Ab2 immunoprecipitatedproMyostatin from muscle extracts. In contrast, AbMyo efficiently boundto latent Myostatin and partially processed precursors in serum, with nodetectable interactions with proMyostatin in the muscle. Given that themuscle is the site where myostatin signaling occurs, this could provideimportant advantages to the Ab2 mechanism of action.

Homogenized muscle lysate was prepared as follows: frozen mousequadriceps were pulverized using a CryoPrep pulverizer (Covaris, WoburnMass.). The pulverized muscle was then resuspended to a concentration of50 mg/mL in M-Per buffer (ThermoFisher Scientific) with 1× Halt™Protease and Phosphatase Inhibitor Cocktail without EDTA (ThermoFisherScientific) The tissue was then crushed using a plastic pestle,(Bio-Plas Cat #4030-PB) and homogenized further with repeated pipettingwith a cut-off pipette tip. Muscle samples were then incubated 30minutes at 4 C with end-over-end rotation. Finally, samples werecentrifuged at 16,100 g for 10 minutes to pellet the insoluble fraction.The soluble fraction was aspirated off and used in downstreamexperiments.

For immunoprecipitation, Ab2, IgG Ctl, or AbMyo antibodies werecovalently conjugated to agarose beads using the Thermo ScientificPierce™ Co-Immunoprecipitation Kit according to the manufacturer'sspecifications. 75 ug of each antibody was conjugated to 50 uL of beadslurry, and 30 ug of antibody was utilized in each immunoprecipitation.The immunoprecipitation was performed against 3 mL of pooled normalmouse serum (Bioreclamation) or 1.05 mL of homogenized soluble mousequadriceps prepared as described above. Antibody conjugated beads andsamples were incubated at 4 C with end-over-end rocking overnight. Afterincubation, the beads were recovered by passing the entire sample volumethrough the spin filters included in the co-immunoprecipitation kitusing the QIAvac 24 Plus vacuum manifold. (Qiagen) The beads were thenwashed 3× with 200 uL of IP lysis/wash buffer, and once with 100 μL of1× conditioning buffer according to the specifications of the kit.Elutions were performed with 50 μL of elution buffer for five minutesand were then mixed with 5 μL of 1M Tris, pH 9.5 in the collection tube.

Myostatin species pulled down by the test antibodies were visualized byWestern blotting utilizing AF1539, (R&D systems) ab124721, (Abcam) AlexaFluor® 680 AffiniPure Donkey Anti-Sheep IgG (H+L), (JacksonImmunoResearch) and IRDye® 800CW Donkey anti-Rabbit IgG (H+L) (LI-CORBiosciences) Thermo Scientific. SEA BLOCK blocking buffer was utilizedfor the blocking and primary antibody incubations.

Example 6: Increased Muscle Mass and Altered Myostatin ProteinExpression in Rats Treated with Ab2 Study Design

Seven to eight week old female Sprague-Dawley rats were administered asingle intravenous dose of either Ab2 (10 mg/kg), a nonfunctional humanIgG control antibody (10 mg/kg), or an equivalent volume of phosphatebuffered saline (PBS). During the course of the study, serum wascollected from 3 rats per group at 4 hours, 48 hours, 7 days, 14 days,21 days, and 28 days after dosing. Collection was done by standardmethods and samples were stored at −80° C. Lean mass was measured byquantitative nuclear magnetic resonance (qNMR) at baseline (prior to day0 dosing) and on days 7, 14, 21, and 28 (8 rats/group) and skeletalmuscles (rectus femoris, tibialis anterior, and soleus) were collectedat the end of study (day 28), weighed, and flash frozen in liquidnitrogen for storage at −80° C.

Results

Drug exposure was measured in serum samples using an ELISA specific tohuman IgG with known quantities of each drug used as a referencestandard. As shown in FIG. 29, 4 hours after injection, both Ab2 and theIgG control antibody are detected in rat serum. As the study progresses,Ab2 exhibits elevated circulating drug levels compared to the IgGcontrol, with an average of 17.1 μg/ml drug in serum at the end of thestudy.

Pharmacodynamic effects of Ab2 treatment were assessed both by measuringlean mass (by qNMR) during the course of the study and by determiningthe weights of dissected muscle at the end of the study. FIG. 30A showslean mass measurements during the course of the study, where ratstreated with Ab2 demonstrate a clear increase in lean mass compared torats treated with PBS or with human IgG Control antibody. Muscle masswas measured by collecting and weighing whole skeletal muscles at theend of the study (28 days). As shown in FIG. 30B, rats treated with Ab2show an increase of 14% and 11% in rectus femoris and tibialis anteriormuscle masses, respectively. Together, these data indicate thattreatment of rats with a single dose of Ab2 leads to long-lastingincreases in muscle mass.

Relative levels of pro and latent Myostatin was determined byquantitative western blotting of muscle lysate or serum samples. Musclelysates were generated from flash frozen muscle samples bypulverization, followed by lysis in T-PER buffer supplemented withprotease and phosphatase inhibitors. After lysis, samples containingequal concentrations of protein were separated by PAGE gels and Westernblotted onto low fluorescence PVDF membrane. For muscle lysates, 10-50ng total protein was loaded onto the gel. Plasma was diluted 1:10 in PBSand 10 μl of each sample loaded onto the gel. As size standards, 0.1-1ng recombinant pro and/or latent Myostatin were also loaded onto thegel. Identification of Myostatin protein was accomplished using anantibody recognizing the prodomain of latent Myostatin (AF1539, R&DSystems), followed by detection with a fluorescently labeled secondaryantibody. For all western blot analyses, a minimum of three samples pergroup were assayed.

Treatment with Ab2 increases latent myostatin levels in rat serum by˜20-fold (FIG. 31A) compared to IgG control-treated rats. These data areconsistent with effects seen with other antibody drugs, reflectingbinding of the drug target in circulation. In rat muscle (rectusfemoris), Ab2 treatment leads to a 1.9× increase (vs. IgGcontrol-treated rats) in the latent form of Myostatin. No statisticallysignificant change in proMyostatin is observed. These data indicate thatAb2 binds its target, pro/latent Myostatin, and alters Myostatinprocessing in muscle as well as in circulation. It was also observedthat Ab2 treatment increases latent, but not proMyostatin in Rat muscle(FIG. 31B).

Example 7: Increased Muscle Mass and Alteration of Myostatin ProteinExpression in Mice Treated with Ab2 and Comparison to a Comparator AntiMyostatin Antibody Study Design

Ten week old male SCID mice were administered a single intraperitonealdose (5 mg/kg) of either Ab2, a nonfunctional human IgG controlantibody, or a comparator antibody (AbMyo) that acts by blocking theMyostatin/receptor interaction. During the course of the study, serumand skeletal muscle were collected at 1 hour, 4 hours, 48 hours, 7 days,14 days, 21 days, 28 days, and 56 days after dosing. Serum collectionwas done by standard methods and samples were stored at −80° C. Skeletalmuscles (rectus femoris, tibialis anterior, and soleus) were collected,weighed, and flash frozen in liquid nitrogen for storage at −80° C. Leanmass was measured by quantitative nuclear magnetic resonance (qNMR) atbaseline (prior to day 0 dosing) and weekly throughout the course of thestudy.

Results

Pharmacodynamic effects of Ab2 treatment were assessed by measuring leanmass (by qNMR) during the course of the study. FIG. 32 shows lean massmeasurements during the course of the study, where mice treated with Ab2demonstrate a clear increase in lean mass compared to mice treated withhuman IgG Control antibody. For the first three weeks of the study, micetreated with the comparator antibody (AbMyo) show increases in lean massequivalent to those in the Ab2 group. However, by 28 days after dosing,AbMyo-treated mice do not maintain increased lean mass. In contrast,mice in the Ab2-treated group maintain their increased lean massthroughout the duration of the study (56 days). These data suggest thatAb2 has a longer duration of action that AbMyo.

Drug exposure was measured in serum samples using an ELISA specific tohuman IgG with known quantities of each drug used as a referencestandard. As shown in FIG. 33, as early as 1 hour after injection, bothAb2 and the comparator antibody (AbMyo) are detected in serum andlevels >1 μg/ml of both antibodies can be detected throughout the study.However, Ab2 exhibits a significantly longer half-life and inferred areaunder the curve (AUCINF) than AbMyo, suggesting that, at similar doses,Ab2 exhibits significantly greater exposure than AbMyo.

Relative levels of pro and latent Myostatin was determined byquantitative western blotting of muscle lysate or serum samples. Musclelysates were generated from flash frozen muscle samples bypulverization, followed by lysis in T-PER buffer supplemented withprotease and phosphatase inhibitors. After lysis, samples containingequal concentrations of protein were separated by PAGE gels and Westernblotted onto low fluorescence PVDF membrane. For muscle lysates, 10-50ng total protein was loaded onto the gel. Plasma was diluted 1:10 in PBSand 10 μl of each sample loaded onto the gel. As size standards, 0.1-1ng recombinant pro and/or latent Myostatin were also loaded onto thegel. Identification of Myostatin protein was accomplished using anantibody recognizing the prodomain of latent Myostatin (AF1539, R&DSystems), followed by detection with a fluorescently labeled secondaryantibody. For all western blot analyses, a minimum of three samples pergroup were assayed.

Serum Myostatin was measured in drug treated mice and in controls usingfluorescent western blotting. Despite the increased serum exposure ofAb2, serum latent Myostatin levels in both Ab2- and AbMyo-treated micewere similar (FIG. 34). These data suggest that the circulating levelsof free drug (not bound to target) are sufficiently greater than thelevel of target, such that the increased serum exposure of Ab2 does nottranslate to larger increases in circulating latent Myostatin than thoseobserved with AbMyo.

Myostatin levels in muscle (rectus femoris) were also evaluated byfluorescent western blotting. Relative levels of latent and proMyostatinwere measured in mouse muscle lysates by fluorescent western blot.Latent Myostatin is elevated in both Ab2 and AbMyo treated muscles (FIG.35A). However, elevation of latent Myostatin in AbMyo-treated musclesreturns to baseline by day 28, while those in Ab2 treated muscles remainelevated until at least this time (P<0.003 vs. AbMyo treatment). Asimilar trend is observed with proMyostatin (FIG. 35B), though thedifference is not statistically significant (P=0.068). These datasuggest a longer duration of action of Ab2 at site of drug action, theskeletal muscle.

Example 8: Ab2 Increases Muscle Force Generation

In this Example, the effects of Ab2 on muscle force generation wereevaluated. Briefly, male C57BL/6J mice were intraperitoneallyadministered IgG (20 mg/kg), Ab2 having a constant region of mouse IgG1isotype (20 mg/kg), or PBS once per week for 4 weeks (n=10 per group).

At study termination, muscles were dissected and weighted, and in vitromuscle performance of the Extensor Digitorum Longus (EDL) muscle wasmeasured in vitro with a 305 C muscle lever system (Aurora ScientificInc., Aurora, CAN) adapted with a horizontal perfusion bath. The musclewas placed in an ice-cold physiological buffered solution and a silksuture tied to the proximal tendon. The muscle was placed in thehorizontal bath of the 305 C muscle lever system and perfused withphysiological buffer oxygenated with 95% O₂/5% CO₂ and kept at 37° C.

Sutures were tied to a fixed post on one side, and the lever arm on theother. A series of 1 Hz and 100 Hz field stimulations (0.2 ms pulse, 100ms duration) at 0.01 Hz frequency were delivered via platinum electrodesflanking the muscle to ensure that the sutures are tight and that themaximal developed force is stable. Once stable, direct musclestimulation—force vs. frequency was measured. Platinum wire electrodesare placed proximal and distal to the muscle belly.

Twitch tension was monitored with a 1 ms pulse and voltage increaseduntil maximal force is achieved. A series of stimulations were thenperformed at increasing frequency of stimulation (1 ms pulse, 250 mstrain duration): 1, 10, 20, 40, 60, 80, 100, 150 Hz, followed by a finalstimulation at 1 Hz.

As depicted in FIG. 36A, following 4 weeks of treatment with Ab2, musclemass and function was increased. Mean EDL weight increased by 33%, andmean gastrocnemius and quadriceps weights increased by 19%.

As depicted in FIG. 36B, maximal force generation increased by 30%following 4 weekly doses of Ab2.

While several embodiments of the present disclosure have been describedand illustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and/or structures for performing thefunctions and/or obtaining the results and/or one or more of theadvantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the presentdisclosure. More generally, those skilled in the art will readilyappreciate that all parameters, dimensions, materials, andconfigurations described herein are meant to be exemplary and that theactual parameters, dimensions, materials, and/or configurations willdepend upon the specific application or applications for which theteachings of the present disclosure is/are used. Those skilled in theart will recognize, or be able to ascertain using no more than routineexperimentation, many equivalents to the specific embodiments of thedisclosure described herein. It is, therefore, to be understood that theforegoing embodiments are presented by way of example only and that,within the scope of the appended claims and equivalents thereto, thedisclosure may be practiced otherwise than as specifically described andclaimed. The present disclosure is directed to each individual feature,system, article, material, and/or method described herein. In addition,any combination of two or more such features, systems, articles,materials, and/or methods, if such features, systems, articles,materials, and/or methods are not mutually inconsistent, is includedwithin the scope of the present disclosure.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Other elements may optionallybe present other than the elements specifically identified by the“and/or” clause, whether related or unrelated to those elementsspecifically identified unless clearly indicated to the contrary. Thus,as a non-limiting example, a reference to “A and/or B,” when used inconjunction with open-ended language such as “comprising” can refer, inone embodiment, to A without B (optionally including elements other thanB); in another embodiment, to B without A (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of” or, when used inthe claims, “consisting of” will refer to the inclusion of exactly oneelement of a number or list of elements. In general, the term “or” asused herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of” “only one of” or“exactly one of” “Consisting essentially of” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” and the like are to be understoodto be open-ended, i.e., to mean including but not limited to. Only thetransitional phrases “consisting of” and “consisting essentially of”shall be closed or semi-closed transitional phrases, respectively, asset forth in the United States Patent Office Manual of Patent ExaminingProcedures, Section 2111.03.

Use of ordinal terms such as “first,” “second,” “third,” etc., in theclaims to modify a claim element does not by itself connote anypriority, precedence, or order of one claim element over another or thetemporal order in which acts of a method are performed, but are usedmerely as labels to distinguish one claim element having a certain namefrom another element having a same name (but for use of the ordinalterm) to distinguish the claim elements.

1-42. (canceled)
 43. A method of preventing muscle loss and/or reducingmuscle atrophy in a subject, the method comprising administering to asubject in need thereof an effective amount of an antibody thatspecifically binds pro/latent myostatin, wherein the antibody comprisesa heavy chain comprising an amino acid sequence of SEQ ID NO:50 and alight chain comprising an amino acid sequence of SEQ ID NO:51.
 44. Themethod of claim 43, wherein the muscle loss and/or muscle atrophy isassociated with a primary myopathy, a secondary myopathy, amyotrophiclateral sclerosis, spinal muscular atrophy (SMA), androgen deficiency, adisease or condition related to aging, a metabolic myopathy, an acquiredmyopathy, a congenital myopathy, a muscular dystrophy, disuse atrophy,and/or cachexia.
 45. The method of claim 44, wherein the primarymyopathy comprises disuse atrophy; the secondary myopathy comprisesdenervation, genetic muscle weakness, or cachexia; the disease orcondition related to aging comprises sarcopenia, frailty, or androgendeficiency; the metabolic myopathy comprises a glycogen storage diseaseor a lipid storage disorder; the acquired myopathy comprises externalsubstance-induced myopathy, myositis, myositis ossificans,rhabdomyolysis, myoglobinuria, or disuse atrophy; the congenitalmyopathy comprises X-linked myotubular myopathy, autosomal dominantcentronuclear myopathy, autosomal recessive centronuclear myopathy,nemaline myopathy, or congenital fiber-type disproportion myopathy; themuscular dystrophy comprises Duchenne's muscular dystrophy, Becker'smuscular dystrophy, facioscapulohumeral (FSH) muscular dystrophy, orLimb-Girdle muscular dystrophy; the disuse atrophy is associated withtrauma, hip fracture, elective joint replacement, critical caremyopathy, spinal cord injury, or stroke; and/or the cachexia isassociated with renal failure, acquired immune deficiency syndrome(AIDS), a cardiac condition, cancer, or aging.
 46. The method of claim43, wherein the muscle loss and/or muscle atrophy is associated withSMA.
 47. The method of claim 43, wherein the muscle loss and/or muscleatrophy is associated with a muscular dystrophy.
 48. The method of claim47, wherein the muscular dystrophy comprises Duchenne's musculardystrophy or Becker's muscular dystrophy.
 49. The method of claim 43,wherein the amount is between about 0.3 mg/kg and about 30 mg/kg perdose.
 50. The method of claim 43, wherein the antibody is administeredto the subject intravenously or subcutaneously.
 51. A method of treatinga subject having, or at risk of developing, spinal muscular atrophy(SMA), the method comprising administering to the subject an effectiveamount of an antibody that specifically binds pro/latent myostatin,wherein the antibody comprises a heavy chain comprising an amino acidsequence of SEQ ID NO:50 and a light chain comprising an amino acidsequence of SEQ ID NO:51.
 52. The method of claim 51, wherein the amountis between about 0.3 mg/kg and about 30 mg/kg per dose.
 53. The methodof claim 51, wherein the amount is effective to increase muscle mass,enhance force generation, prevent muscle loss, and/or reduce muscleatrophy in the subject.
 54. The method of claim 51, wherein the antibodyis administered to the subject intravenously or subcutaneously.